Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients

Abstract

Objectives: Elevated circulating DNA (cirDNA) concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release. We carried out the first prospective, multicenter study of the dynamics of cirDNA and neutrophil extracellular trap (NETs) markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients.

Methods: We examined the plasma levels of two NETs protein markers [myeloperoxidase (MPO) and neutrophil elastase (NE)], as well as levels of cirDNA of nuclear (cir-nDNA) and mitochondrial (cir-mtDNA) origin in 29 colon, prostate, and breast cancer patients and in 114 healthy individuals (HI).

Results: The synergistic analytical information provided by these markers revealed that: (i) NETs formation contributes to post-surgery conditions; (ii) post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer [r = 0.60 (P < 0.001) and r = 0.53 (P < 0.01), respectively], but not in prostate and breast cancer; (iii) each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics, but overall the pre-and post-surgery median values of cir-nDNA, NE, and MPO were significantly higher in cancer patients than in HI.

Conclusion: Taken as a whole, our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period, depending on surgical procedure or cancer type. By contrast, cir-mtDNA is poorly associated with NETs formation in the studied perioperative period, which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

Keywords

circulating DNA / perioperative period / neutrophil extracellular traps / neutrophil elastase / myeloperoxidase / minimal residual disease

Acknowledgements

This study is funded by grant NIMAO 2016-08 and partially supported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553 and the “Société Française des acides nucléiques circulants” (SFAC). The promotor of this study is the Centre Hospitalier Universitaire de Nîmes. The authors thank Cormac Mc Carthy (Mc Carthy Consultant, Montpellier) for English editing (financial compensation). We thank our patients and their families for their trust; and all the participating physicians and supporting staff. We thank all healthy donors who participated in this study. We also thank the clinical investigators of the centers who participated in this study.

Author contributions

Conceptualization and Funding acquisition: P.C. and A.R.T. Methodology: A.K., B.P., and A.R.T. Investigation: A.K.,B.P., E.P., A.M., Y.G., X.C. Supervision: A.R.T. Formal Analysis: A.K. and B.P. Writing - original draft, review & editing: A.K., B.P., P.C. and A.R.T. All of the authors discussed the results and approved the manuscript.

Conflict of interest

None declared. As an Editorial Board Member of Precision Clinical Medicine, the corresponding author Alain R. Thierry was blinded from reviewing and making decisions on this manuscript.

References

[1] Bronkhorst AJ, Ungerer V, Diehl F et al. Towards systematic nomenclature for cell-free DNA. Hum Genet 2021;140:565-78. https://doi.org/10.1007/s00439-020-02227-2.

[2] Diaz LA, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 2014;32:579-86. https://doi.org/10.1200/JC O.2012.45.2011.

[3] Thierry AR, El Messaoudi S, Gahan PB et al. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev 2016;35:347-76. https://doi.org/10.1007/s10555-016-9629-x.

[4] Wan JCM, Massie C, Garcia-Corbacho J et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 2017;17:223-38. https://doi.org/10.1038/nrc.2017.7.

[5] Benešová L, Hálková T, Ptáčková R et al. Significance of postoperative follow-up of patients with metastatic colorectal cancer using circulating tumor DNA. World J Gastroenterol 2019;25:693948. https://doi.org/10.3748/wjg.v25.i48.6939.

[6] Parikh AR, Van Seventer EE, Siravegna G et al. Minimal residual disease detection using a plasma-only circulating tumor DNA assay in patients with colorectal cancer. Clin Cancer Res 2021;27:5586-94. https://doi.org/10.1158/1078-0432.CCR-21-0410.

[7] Tie J, Cohen JD, Wang Y et al. Circulating tumor DNA analyses as markers of recurrence risk and benefit of adjuvant therapy for stage III colon cancer. JAMA Oncol 2019;5:1710-7. https://doi.org/ 10.1001/jamaoncol.2019.3616.

[8] Sefrioui D, Beaussire L, Gillibert A et al. CEA, CA19-9, circulating DNA and circulating tumour cell kinetics in patients treated for metastatic colorectal cancer (mCRC). Br J Cancer 2021;125:72533. https://doi.org/10.1038/s41416-021-01431-9.

[9] Thierry AR, Pastor B, Jiang Z-Q et al. Circulating DNA demonstrates convergent evolution and common resistance mechanisms during treatment of colorectal cancer. Clin Cancer Res 2017;23:4578-91. https://doi.org/10.1158/1078-0432.CCR-17-023 2.

[10] Cohen JD, Li L, Wang Y et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 2018;359:926-30. https://doi.org/10.1126/science.aar3247.

[11] Cristiano S, Leal A, Phallen J et al. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature 2019;570:385-9. https://doi.org/10.1038/s41586-019-1272-6.

[12] Sanchez C, Roch B, Mazard T et al. Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics. JCI Insight 2021;6:144561. https://doi.org/10.1172/jci. insight.144561.

[13] Tanos R, Tosato G, Otandault A et al. Machine learning-assisted evaluation of circulating DNA quantitative analysis for cancer screening. Adu Sci (Weinh) 2020;7:2000486. https://doi.org/10.100 2/advs. 202000486.

[14] Margraf S, Lögters T, Reipen J et al. Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis. Shock 2008;30:352-8. https://doi.org/10.1097/SHK.0b013e31816a 6bb1.

[15] Qi Y, Uchida T, Yamamoto M et al. Perioperative elevation in cellfree DNA levels in patients undergoing cardiac surgery: possible contribution of neutrophil extracellular traps to Perioperative renal dysfunction. Anesthesiology Research and Practice 2016;2016:111. https://doi.org/10.1155/2016/2794364.

[16] Onuma AE, Zhang H, Gil L et al. Surgical stress promotes tumor progression: A focus on the impact of the immune response. JCM 2020;9:4096. https://doi.org/10.3390/jcm9124096.

[17] Cheng H, Chen BP-H, Soleas IM et al. Prolonged operative duration increases risk of surgical site infections: A systematic review. Surgical Infections 2017;18:722-35. https://doi.org/10.1089/sur.2017.089.

[18] Kim R. Anesthetic technique and cancer recurrence in oncologic surgery: unraveling the puzzle. Cancer Metastasis Rev 2017;36:159-77. https://doi.org/10.1007/s10555-016-9647-8.

[19] Lo YM, Rainer TH, Chan LY et al. Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000;46:319-23. https://doi.org/10.1093/clinchem/46.3.319.

[20] Henriksen TV, Reinert T, Christensen E et al. The effect of surgical trauma on circulating free DNA levels in cancer patientsImplications for studies of circulating tumor DNA. Molecular Oncology 2020;14:1670-9. https://doi.org/10.1002/1878-0261.12729.

[21] Al Amir Dache Z, Thierry AR. Mitochondria-derived cell-to-cell communication. Cell Reports 2023;42:112728. https://doi.org/10.1016/j.celrep.2023.112728.

[22] Picca A, Lezza AMS, Leeuwenburgh C et al. Circulating mitochondrial DNA at the crossroads of mitochondrial dysfunction and inflammation during aging and muscle wasting disorders. Rejuvenation Research 2018;21:350-9. https://doi.org/10.1089/rej.2017.1989.

[23] Urban CF, Lourido S, Zychlinsky A. How do microbes evade neutrophil killing? Cell Microbiol 2006;8:1687-96. https://doi.org/10.1 111/j.1462-5822.2006.00792.x.

[24] Waisberg M, Molina-Cruz A, Mizurini DM et al. Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis. PLoS Pathog 2014;10:e1004338. https://doi.org/10.1371/journal.ppat.1004338.

[25] Yipp BG, Petri B, Salina D et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 2012;18:1386-93. https://doi.org/10.1038/nm.2847.

[26] Brinkmann V. Neutrophil extracellular traps in the second decade. J Innate Immun 2018;10:414-21. https://doi.org/10.1159/000489829.

[27] Mattox AK, Douville C, Wang Y et al. The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer. Cancer Discovery 2023;10:2166-2179. https://doi.org/10.1158/2159-8290.CD-21-1252

[28] Brinkmann V, Reichard U, Goosmann C et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532-5. https://doi.org/10.1126/science. 1092385.

[29] Metzler KD, Goosmann C, Lubojemska A et al. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis. Cell Reports 2014;8:883-96. https://doi.org/10.1016/j.celrep.2014.06.044.

[30] Cools-Lartigue J, Spicer J, McDonald B et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest 2013;123:3446-58. https://doi.org/10.1172/JCI67484.

[31] Park J, Wysocki RW, Amoozgar Z et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Sci Transl Med 2016;8:361ra138. https://doi.org/10.1126/scitranslmed.aag1711.

[32] Tohme S, Simmons RL, Tsung A. Surgery for cancer: A trigger for metastases. Cancer Research. 2017; 77:1548-52. https://doi.org/10.1158/0008-5472.CAN-16-1536.

[33] Tohme S, Yazdani HO, Al-Khafaji AB et al. Neutrophil extracellular traps promote the development and progression of liver metastases after surgical stress. Cancer Research 2016;76:1367-80. https://doi.org/10.1158/0008-5472.CAN-15-1591.

[34] Gentles AJ, Newman AM, Liu CL et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 2015;21:938-45. https://doi.org/10.1038/nm.3909.

[35] Templeton AJ, McNamara MG, Šeruga B et al. Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis. J Natl Cancer Inst 2014;106:dju124. https://doi.org/10.1093/jnci/dju124.

[36] Kos K, de Visser KE. Neutrophils create a fertile soil for metastasis. Cancer Cell 2021;39:301-3. https://doi.org/10.1016/j.ccell.2021.01.009.

[37] Nolan E, Malanchi I. Neutrophil "safety net" causes cancer cells to metastasize and proliferate. Nature 2020;583:32-3. https://doi.org/10.1038/d41586-020-01672-3.

[38] Wang J. Neutrophils in tissue injury and repair. Cell Tissue Res 2018;371:531-9. https://doi.org/10.1007/s00441-017-2785-7.

[39] Kurosawa S, Kato M. Anesthetics, immune cells, and immune responses. J Anesth 2008;22:263-77. https://doi.org/10.1007/s00540-008-0626-2.

[40] Saithong S, Saisorn W, Tovichayathamrong P et al. Antiinflammatory effects and decreased formation of neutrophil extracellular traps by Enoxaparin in COVID-19 patients. IJMS 2022;23:4805. https://doi.org/10.3390/ijms23094805.

[41] Pastor B, Abraham J-D, Pisareva E et al. Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer. iScience 2022;25:103826. https://doi.org/10.1016/j.isci.2022.103826.

[42] Pisareva E, Mihalovičová L, Pastor B et al. Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA. Genome Med 2022;14:135. https://doi.org/10.1186/s13073-022-01125-8.

[43] Aarab Y, Ramin S, Odonnat T et al. Pectoral nerve blocks for breast augmentation surgery: A randomized, double-blind, dualcentered controlled trial. Anesthesiology 2021;135:442-53. https://doi.org/10.1097/ALN.0000000000003855.

[44] Meddeb R, Pisareva E, Thierry AR. Guidelines for the preanalytical conditions for analyzing circulating cell-free DNA. Clin Chem 2019;65:623-33. https://doi.org/10.1373/clinchem.2018.298323.

[45] Sorber L, Zwaenepoel K, Deschoolmeester V et al. A comparison of cell-free DNA isolation kits: isolation and quantification of cell-free DNA in plasma. J Mol Diagn 2017;19:162-8. https://doi.org/10.1016/j.jmoldx.2016.09.009.

[46] Mouliere F, El Messaoudi S, Pang D et al. Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer. Mol Oncol 2014;8:927-41. https://doi.org/10.1016/j.molonc.2014.02.005.

[47] Thierry AR, Mouliere F, El Messaoudi S et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 2014;20:430-5. https://doi.org/10.1038/nm.3511.

[48] Thierry AR, El Messaoudi S, Mollevi C et al. Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment. Ann Oncol 2017;28:2149-59. https://doi.org/10.1093/annonc/mdx330.

[49] Al Amir Dache Z, Otandault A, Tanos R, et al. Blood contains circulating cell-free respiratory competent mitochondria. FASEB J 2020;3:3616-3630. https://doi.org/10.1096/fj.201901917RR

[50] Bustin SA, Benes V, Garson JA et al. The MIQE Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 2009;55:611-22. https://doi.org/10.1373/clinchem.2008.112797.

[51] Pisareva E, Roch B, Sanchez C et al. Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA. Front Genet 2023;14:1104732. https://doi.org/10.3389/fgene.2023.1104732.

[52] Sanchez C, Roch B, Mazard T et al. Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics. JCI Insight 2021;6:e144561. https://doi.org/10.1172/jci.insight.144561.

[53] Pisareva E, Badiou S, Mihalovičová L et al. Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients. Journal of Medical Virology 2023;95:e28209. https://doi.org/10.1002/jmv.28209.

[54] Diehl F, Li M, Dressman D et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. PNAS 2005;102:16368-73. https://doi.org/10.1073/pnas.0507904102.

[55] Mouliere F, Robert B, Arnau Peyrotte E et al. High fragmentation characterizes tumour-derived circulating DNA. PLoS ONE 2011;6:e23418. https://doi.org/10.1371/journal.pone.0023418.

[56] Haller N, Helmig S, Taenny P et al. Circulating, cell-free DNA as a marker for exercise load in intermittent sports. PLoS ONE 2018;13:e0191915. https://doi.org/10.1371/journal.pone.0191915.

[57] Thierry AR, Roch B. Neutrophil extracellular traps and byproducts play a key role in COVID-19: pathogenesis, risk factors, and therapy. JCM 2020;9:2942. https://doi.org/10.3390/jcm9092942.

[58] Tie J, Cohen JD, Wang Y et al. Circulating tumor DNA analyses as markers of recurrence risk and benefit of adjuvant therapy for stage III colon cancer. JAMA Oncol 2019;5:1710. https://doi.org/10.1001/jamaoncol.2019.3616.

[59] Ma Y, Gan J, Bai Y et al. Minimal residual disease in solid tumors: an overview. Front Med 2023;17:649-74.

[60] Fleming CA, O'Leary DP, Wang J et al. Association of observed perioperative cell-free DNA dynamics with early recurrence in patients with colon cancer. JAMA Surg 2020;1:168-70. https://doi.org/10.1001/jamasurg.2019.4706

[61] Le-Wendling L, Nin O, Capdevila X. Cancer recurrence and regional anesthesia: the theories, the data, and the future in outcomes. Pain Med 2016;17:756-75.

[62] Wei Z, Shah N, Deng C et al. Circulating DNA addresses cancer monitoring in non small cell lung cancer patients for detection and capturing the dynamic changes of the disease. SpringerPlus 2016;5:531. https://doi.org/10.1186/s40064-016-2141-5.

[63] Snyder GL, Greenberg S. Effect of anaesthetic technique and other perioperative factors on cancer recurrence. Br J Anaesth 2010;105:106-15. https://doi.org/10.1093/bja/aeq164.

[64] Van Der Bij GJ, Oosterling SJ, Beelen RHJ et al. The perioperative period is an underutilized window of therapeutic opportunity in patients with colorectal cancer. Ann Surg 2009;249:727-34. https://doi.org/10.1097/SLA.0b013e3181a3ddbd.

[65] Seth R, Tai L-H, Falls T et al. Surgical stress promotes the development of cancer metastases by a coagulation-dependent mechanism involving natural killer cells in a murine model. Ann Surg 2013;258:158-68. https://doi.org/10.1097/SLA.0b013e31826fcbdb.

[66] Palumbo JS, Talmage KE, Massari JV et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 2005;105:178-85. https://doi.org/10.1182/blood-2004-06-2272.

[67] Chen J, Deng Q, Pan Y et al. Prognostic value of neutrophil-tolymphocyte ratio in breast cancer. FEBS Open Bio 2015;5:502-7. https://doi.org/10.1016/j.fob.2015.05.003.

[68] Guthrie GJK, Charles KA, Roxburgh CSD et al. The systemic inflammation-based neutrophil-lymphocyte ratio: experience in patients with cancer. Crit Rev Oncol Hematol 2013;88:218-30. https://doi.org/10.1016/j.critrevonc.2013.03.010.

[69] Moss J, Magenheim J, Neiman D et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cellfree DNA in health and disease. Nat Comm 2018;9:5068. https://doi.org/10.1038/s41467-018-07466-6

[70] Loyfer N, Magenheim J, Peretz A et al. A DNA methylation atlas of normal human cell types. Nature 2023;613:355-64. https://doi.org/10.1038/s41586-022-05580-6.

[71] Leal A, Van Grieken NCT, Palsgrove DN et al. White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer. Nat Commun 2020;11:525. https://doi.org/10.1038/s41467-020-14310-3.

[72] Benhaim L, Bouché O, Normand C et al. Circulating tumor DNA is a prognostic marker of tumor recurrence in stage II and III colorectal cancer: multicentric, prospective cohort study (ALGECOLS). Europ J Cancer 2021;159:24-33. https://doi.org/10.1016/j.ejca.2021.09.004.

[73] Ohara S, Suda K, Sakai K et al. Prognostic implications of preoperative versus postoperative circulating tumor DNA in surgically resected lung cancer patients: a pilot study. Transl Lung Cancer Res 2020;9:1915-23. https://doi.org/10.21037/tlcr-20-505.

[74] Jorch SK, Kubes P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med 2017;23:279-87. https://doi.org/10.1038/nm.4294.

[75] Rossaint R, Afshari A, Bouillon B et al. The european guideline on management of major bleeding and coagulopathy following trauma: sixth edition. Crit Care 2023;27:80. https://doi.org/10.1186/s13054-023-04327-7.

[76] Wu H, Wang S, Lv H et al. Effect of thoracic epidural anesthesia on perioperative neutrophil extracellular trapping markers in patients undergoing anesthesia and surgery for colorectal cancer: A randomized, controlled trial. Ann Surg Oncol 2023; 12:75617568. https://link.springer.com/10.1245/s10434-023-14077-z

[77] Jahr S, Hentze H, Englisch S et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 2001;61:165965.

[78] Holdenrieder S, Mueller S, Stieber P. Stability of nucleosomal DNA fragments in serum. Clin Chem 2005;51:1026-9. https://doi.org/10.1373/clinchem.2005.048454.

[79] Otandault A, Abraham J-D, Al Amir Dache Z et al. Hypoxia differently modulates the release of mitochondrial and nuclear DNA. Br J Cancer 2020;122:715-25. https://doi.org/10.1038/s41416-019-0716-y.

[80] Thierry AR, Pisareva E. A new paradigm of the origins of circulating DNA in patients with cancer. Cancer Discov 2023;13:2122-4. https://doi.org/10.1158/2159-8290.CD-23-0824.