Breast Cancer Research and Treatment

, Volume 169, Issue 1, pp 69–82 | Cite as

Circulating free DNA integrity and concentration as independent prognostic markers in metastatic breast cancer

  • Jie Cheng
  • Tim Holland-Letz
  • Markus Wallwiener
  • Harald Surowy
  • Katarina Cuk
  • Sarah Schott
  • Andreas Trumpp
  • Klaus Pantel
  • Christof Sohn
  • Andreas Schneeweiss
  • Barbara Burwinkel
Clinical trial



Non-invasive blood-based molecular markers have been investigated for cancer diagnosis and prognosis. Circulating free or cell-free DNA (cfDNA) variables have been shown to be putative markers in breast cancer prognosis.


Here, we investigated the potential prognostic ability of cfDNA concentration and cfDNA integrity (cfDI) in a study cohort of 268 patients by quantitative PCR. We compared cfDNA concentration and cfDI at baseline and after one cycle of therapy in metastatic breast cancer (MBC) patients.


A significantly increased cfDI (P = 1.21E-7 for ALU and P = 1.87E-3 for LINE1) and decreased cfDNA concentration (P = 1.17E-3 for ALU and P = 1.60E-2 for LINE1) in both repetitive DNA elements after one cycle of therapy was observed. A multiple Cox regression model indicated that cfDI and cfDNA concentration can serve as independent prognostic markers in patients at baseline with HR (95% CI) of 0.70 (0.48–1.01) for ALU cfDI, 0.63 (0.44–0.92) for LINE1 cfDI, 2.44 (1.68–3.53) for ALU cfDNA concentration, and 2.12 (1.47–3.06) for LINE1 cfDNA concentration and after one cycle of therapy with HR (95% CI) of 0.59 (0.42–0.84) for ALU cfDI, 0.51 (0.36–0.74) for LINE1 cfDI, 1.59 (1.31–1.92) for ALU cfDNA concentration, and 1.30 (1.17–1.45) for LINE1 cfDNA concentration, respectively. By comparing integrated prediction error of different models, cfDNA variables were shown to improve the prognostic power of the CTC status.


We hereby show that cfDNA variables, especially in combination with other markers, can serve as attractive prognostic markers for MBC patients at baseline and during the systematic therapy.


Metastatic breast cancer Circulating DNA concentration Circulating DNA integrity Circulating tumor cells Prognostic marker 



Area under the curve




Circulating free or cell-free DNA integrity


Circulating free or cell-free DNA


Confidence interval


Circulating tumor cell


Hazard ratio


Integrated prediction errors


Metastatic breast cancer


National Center for Tumor Diseases, Heidelberg, Germany


Progression-free survival


Overall survival



We thank the study participants and all our colleagues who helped us with patient recruitment, blood collection, and processing. The study has been supported by cellgene.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2018_4666_MOESM1_ESM.docx (1.3 mb)
Supplementary material 1 (DOCX 1312 kb)


  1. 1.
    Siegel RL et al (2016) Cancer statistics, 2016. CA Cancer J Clin 66:7–30CrossRefPubMedGoogle Scholar
  2. 2.
    Weigelt B et al (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602CrossRefPubMedGoogle Scholar
  3. 3.
    Mehlen P et al (2006) Metastasis: a question of life or death. Nat Rev Cancer 6:449–458CrossRefPubMedGoogle Scholar
  4. 4.
    Swain SM et al (2015) Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med 372:724–734CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Walker AJ et al (2016) FDA approval of palbociclib in combination with fulvestrant for the treatment of hormone receptor-positive, HER2-negative metastatic breast cancer. Clin Cancer Res 22:4968CrossRefPubMedGoogle Scholar
  6. 6.
    Schwarzenbach H et al (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11:426–437CrossRefPubMedGoogle Scholar
  7. 7.
    Cuk K et al (2013) Circulating microRNAs in plasma as early detection markers for breast cancer. Int J Cancer 132:1602–1612CrossRefPubMedGoogle Scholar
  8. 8.
    Madhavan D et al (2013) Cancer diagnosis and prognosis decoded by blood-based circulating microRNA signatures. Front Genet 4:116PubMedPubMedCentralGoogle Scholar
  9. 9.
    Cuk K et al (2013) Plasma microRNA panel for minimally invasive detection of breast cancer. PLoS ONE 8:e76729CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Madhavan D et al (2016) Circulating miRNAs with prognostic value in metastatic breast cancer and for early detection of metastasis. Carcinogenesis 37:461–470CrossRefPubMedGoogle Scholar
  11. 11.
    Madhavan D et al (2012) Circulating miRNAs as surrogate markers for circulating tumor cells and prognostic markers in metastatic breast cancer. Clin Cancer Res 18:5972–5982CrossRefPubMedGoogle Scholar
  12. 12.
    Madhavan D et al (2014) Plasma DNA integrity as a biomarker for primary and metastatic breast cancer and potential marker for early diagnosis. Breast Cancer Res Treat 146:163–174CrossRefPubMedGoogle Scholar
  13. 13.
    Wallwiener M et al (2014) Serial enumeration of circulating tumor cells predicts treatment response and prognosis in metastatic breast cancer: a prospective study in 393 patients. BMC Cancer 14:512CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dawson SJ et al (2013) Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368:1199–1209CrossRefPubMedGoogle Scholar
  15. 15.
    Cheng J et al (2017) Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence. Oncotarget 8:54537PubMedPubMedCentralGoogle Scholar
  16. 16.
    Schwarzenbach H et al (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11:426–437CrossRefPubMedGoogle Scholar
  17. 17.
    Diaz LA Jr et al (2014) Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 32:579–586CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hao TB et al (2014) Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer. Br J Cancer 111:1482–1489CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Szpechcinski A et al (2015) Cell-free DNA levels in plasma of patients with non-small-cell lung cancer and inflammatory lung disease. Br J Cancer 113:476–483CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Huang ZH et al (2006) Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett 243:64–70CrossRefPubMedGoogle Scholar
  21. 21.
    Jiang WW et al (2006) Increased plasma DNA integrity index in head and neck cancer patients. Int J Cancer 119:2673–2676CrossRefPubMedGoogle Scholar
  22. 22.
    Wang BG et al (2003) Increased plasma DNA integrity in cancer patients. Cancer Res 63:3966–3968PubMedGoogle Scholar
  23. 23.
    El-Shazly SF et al (2010) Evaluation of serum DNA integrity as a screening and prognostic tool in patients with hepatitis C virus-related hepatocellular carcinoma. Int J Biol Markers 25:79–86CrossRefPubMedGoogle Scholar
  24. 24.
    Stötzer OJ et al (2014) Diagnostic relevance of plasma DNA and DNA integrity for breast cancer. Tumor Biol 35:1183–1191CrossRefGoogle Scholar
  25. 25.
    McShane LM et al (2005) REporting recommendations for tumour MARKer prognostic studies (REMARK). Br J Cancer 93:387–391CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Riethdorf S et al (2007) Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cell search system. Clin Cancer Res 13:920–928CrossRefPubMedGoogle Scholar
  27. 27.
    Wallwiener M et al (2015) The impact of HER2 phenotype of circulating tumor cells in metastatic breast cancer: a retrospective study in 107 patients. BMC Cancer 15:403CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Cristofanilli M et al (2005) Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 23:1420–1430CrossRefPubMedGoogle Scholar
  29. 29.
    Leon SA et al (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646–650PubMedGoogle Scholar
  30. 30.
    Deligezer U et al (2008) Effect of adjuvant chemotherapy on integrity of free serum DNA in patients with breast cancer. Ann N Y Acad Sci 1137:175–179CrossRefPubMedGoogle Scholar
  31. 31.
    Cheng J et al. (2017) Cell-free circulating DNA Integrity based on peripheral blood as a biomarker for diagnosis of cancer: a systematic review. Cancer Epidemiol BiomarkGoogle Scholar
  32. 32.
    Mead R et al (2011) Circulating tumour markers can define patients with normal colons, benign polyps, and cancers. Br J Cancer 105:239–245CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Huang A et al (2016) Plasma circulating cell-free DNA integrity as a promising biomarker for diagnosis and surveillance in patients with hepatocellular carcinoma. J Cancer 7:1798–1803CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Umetani N et al (2006) Prediction of breast tumor progression by integrity of free circulating DNA in serum. J Clin Oncol 24:4270–4276CrossRefPubMedGoogle Scholar
  35. 35.
    Jahr S et al (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61:1659–1665PubMedGoogle Scholar
  36. 36.
    Heitzer E et al (2015) Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 61:112–123CrossRefPubMedGoogle Scholar
  37. 37.
    Underhill HR et al (2016) Fragment Length of Circulating Tumor DNA. PLoS Genet 12:e1006162CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Jiang P et al (2015) Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci USA 112:E1317–E1325CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Atamaniuk J et al (2004) Increased concentrations of cell-free plasma DNA after exhaustive exercise. Clin Chem 50:1668–1670CrossRefPubMedGoogle Scholar
  40. 40.
    van der Vaart M et al (2007) The origin of circulating free DNA. Clin Chem 53:2215CrossRefPubMedGoogle Scholar
  41. 41.
    Umetani N et al (2006) Prediction of breast tumor progression by integrity of free circulating DNA in serum. J Clin Oncol 24:4270–4276CrossRefPubMedGoogle Scholar
  42. 42.
    Chandarlapaty S et al (2016) Prevalence of ESR1 Mutations in Cell-Free DNA and Outcomes in Metastatic Breast Cancer: a secondary analysis of the BOLERO-2 clinical trial. JAMA Oncol 2:1310–1315CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Budd GT et al (2006) Circulating tumor cells versus imaging—predicting overall survival in metastatic breast cancer. Clin Cancer Res 12:6403CrossRefPubMedGoogle Scholar
  44. 44.
    Kronek L-P et al (2008) Logical analysis of survival data: prognostic survival models by detecting high-degree interactions in right-censored data. Bioinformatics 24:i248–i253CrossRefPubMedGoogle Scholar
  45. 45.
    El Messaoudi S et al (2013) Circulating cell free DNA: preanalytical considerations. Clin Chim Acta 424:222–230CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jie Cheng
    • 1
    • 2
  • Tim Holland-Letz
    • 3
  • Markus Wallwiener
    • 4
    • 5
  • Harald Surowy
    • 1
    • 2
  • Katarina Cuk
    • 1
    • 2
  • Sarah Schott
    • 4
  • Andreas Trumpp
    • 6
    • 7
  • Klaus Pantel
    • 8
  • Christof Sohn
    • 4
  • Andreas Schneeweiss
    • 4
    • 5
  • Barbara Burwinkel
    • 1
    • 2
  1. 1.Division of Molecular EpidemiologyGerman Cancer Research Center (DKFZ)HeidelbergGermany
  2. 2.Molecular Biology of Breast Cancer, Department of Gynecology and ObstetricsUniversity of HeidelbergHeidelbergGermany
  3. 3.Department of BiostatisticsGerman Cancer Research Center (DKFZ)HeidelbergGermany
  4. 4.Department of Gynecology and ObstetricsUniversity Women’s ClinicHeidelbergGermany
  5. 5.National Center for Tumor DiseasesUniversity of HeidelbergHeidelbergGermany
  6. 6.Division of Stem Cells and CancerGerman Cancer Research Center (DKFZ)HeidelbergGermany
  7. 7.Hi-STEM-Heidelberg Institute for Stem Cell Technology and Experimental Medicine, GmbHHeidelbergGermany
  8. 8.Department of Tumor BiologyUniversity Hospital Hamburg-EppendorfHamburgGermany

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