Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

  • Troels BechmannEmail author
  • Rikke Fredslund Andersen
  • Niels Pallisgaard
  • Jonna Skov Madsen
  • Else Maae
  • Erik Hugger Jakobsen
  • Anne Marie Bak Jylling
  • Karina Dahl Steffensen
  • Anders Jakobsen
Original Paper



Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in cfDNA during neoadjuvant chemotherapy.

Patients and methods

The study included 50 patients from a prospective cohort analyzed during neoadjuvant chemotherapy. Fifty healthy women with no history of cancer served as control group and 15 patients with metastatic breast cancer were used to validate the assay. Total cfDNA and HER2 gene amplification were measured by quantitative real-time polymerase chain reaction.


Plasma HER2 gene copy number (p = 0.794), HER2 gene amplification (p = 0.127) and total cfDNA (p = 0.440) did not differ significantly from the levels in the control group. Eighteen patients (36 %) obtained pathological complete response (pCR). HER2 gene copy number before the operation was significantly higher than the baseline level (p < 0.0001), but there was no difference between patients with and without pCR (p = 0.569). Likewise, there was no difference in plasma HER2 gene amplification between tissue HER2-positive and -negative patients (p = 0.754).


The results indicate that neither total cfDNA nor HER2 gene copy number is elevated in primary breast cancer patients compared to healthy controls. The level of both parameters increased during neoadjuvant chemotherapy, but without any relation to treatment effect. There was no indication of plasma HER2 gene amplification in the HER2-positive patients in the neoadjuvant setting.


Breast cancer Cell-free DNA HER2 amplification Neoadjuvant chemotherapy 



The study was financed by the Vejle Hospital Research Foundation. The authors thank Marianne Mose Hansen and Lone Frischknecht for their excellent laboratory work and the Department of Surgery, Vejle Hospital, for the enrollment of patients. Furthermore, we would like to thank research assistant Karin Larsen for proofreading.

Conflict of interest

All authors declare no conflicts of interest.

Supplementary material

432_2013_1413_MOESM1_ESM.doc (28 kb)
Supplementary material 1 (DOC 28 kb)


  1. Andersen RF, Spindler KLG, Jakobsen A, Pallisgaard N (2012) Plasma is superior to serum for cfDNA mutation detection and monitoring. Eur J Cancer 48(suppl. 6): 148–149 Ref Type: AbstractGoogle Scholar
  2. Anker P, Mulcahy H, Chen XQ, Stroun M (1999) Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 18:65–73PubMedCrossRefGoogle Scholar
  3. Bergqvist J, Ohd JF, Smeds J, Klaar S, Isola J, Nordgren H, Elmberger GP, Hellborg H, Bjohle J, Borg AL, Skoog L, Bergh J (2007) Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome. Ann Oncol 18:845–850PubMedCrossRefGoogle Scholar
  4. Catarino R, Ferreira MM, Rodrigues H, Coelho A, Nogal A, Sousa A, Medeiros R (2008) Quantification of free circulating tumor DNA as a diagnostic marker for breast cancer. DNA Cell Biol 27:415–421PubMedCrossRefGoogle Scholar
  5. Egervari K, Toth J, Nemes Z, Szollosi Z (2009) An alternative and reliable real-time quantitative PCR method to determine HER2/neu amplification in breast cancer. Appl Immunohistochem Mol Morphol 17:247–254PubMedCrossRefGoogle Scholar
  6. Fujita N, Nakayama T, Yamamoto N, Kim SJ, Shimazu K, Shimomura A, Maruyama N, Morimoto K, Tamaki Y, Noguchi S (2012) Methylated DNA and total DNA in serum detected by one-step methylation-specific PCR is predictive of poor prognosis for breast cancer patients. Oncology 83:273–282PubMedCrossRefGoogle Scholar
  7. Gal S, Fidler C, Lo YM, Taylor M, Han C, Moore J, Harris AL, Wainscoat JS (2004) Quantitation of circulating DNA in the serum of breast cancer patients by real-time PCR. Br J Cancer 90:1211–1215PubMedCrossRefGoogle Scholar
  8. Galeazzi M, Morozzi G, Piccini M, Chen J, Bellisai F, Fineschi S, Marcolongo R (2003) Dosage and characterization of circulating DNA: present usage and possible applications in systemic autoimmune disorders. Autoimmun Rev 2:50–55PubMedCrossRefGoogle Scholar
  9. Gong B, Xue J, Yu J, Li H, Hu H, Yen H, Hu J, Dong Q, Chen F (2012) Cell-free DNA in blood is a potential diagnostic biomarker of breast cancer. Oncol Lett 3:897–900PubMedGoogle Scholar
  10. Holdenrieder S, Burges A, Reich O, Spelsberg FW, Stieber P (2008) DNA integrity in plasma and serum of patients with malignant and benign diseases. Ann N Y Acad Sci 1137:162–170PubMedCrossRefGoogle Scholar
  11. Huang ZH, Li LH, Hua D (2006) Quantitative analysis of plasma circulating DNA at diagnosis and during follow-up of breast cancer patients. Cancer Lett 243:64–70PubMedCrossRefGoogle Scholar
  12. Koffler D, Agnello V, Winchester R, Kunkel HG (1973) The occurrence of single-stranded DNA in the serum of patients with systemic lupus erythematosus and other diseases. J Clin Invest 52:198–204PubMedCrossRefGoogle Scholar
  13. Kohler C, Radpour R, Barekati Z, Asadollahi R, Bitzer J, Wight E, Burki N, Diesch C, Holzgreve W, Zhong XY (2009) Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors. Mol Cancer 8:105. doi: 10.1186/1476-4598-8-105 PubMedCrossRefGoogle Scholar
  14. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646–650PubMedGoogle Scholar
  15. Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M, Cristofanilli M, Hortobagyi GN, Pusztai L (2008) Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol 26:1275–1281PubMedCrossRefGoogle Scholar
  16. Mandel P, Metais P (1948) Les acides nucleiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil 142:241–243PubMedGoogle Scholar
  17. Page K, Hava N, Ward B, Brown J, Guttery DS, Ruangpratheep C, Blighe K, Sharma A, Walker RA, Coombes RC, Shaw JA (2011) Detection of HER2 amplification in circulating free DNA in patients with breast cancer. Br J Cancer 104:1342–1348PubMedCrossRefGoogle Scholar
  18. Pathak AK, Bhutani M, Kumar S, Mohan A, Guleria R (2006) Circulating cell-free DNA in plasma/serum of lung cancer patients as a potential screening and prognostic tool. Clin Chem 52:1833–1842PubMedGoogle Scholar
  19. Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, Slamon DJ (2000) Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 18:3651–3664PubMedGoogle Scholar
  20. Payne RE, Hava NL, Page K, Blighe K, Ward B, Slade M, Brown J, Guttery DS, Zaidi SA, Stebbing J, Jacob J, Yague E, Shaw JA, Coombes RC (2012) The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy. Br J Cancer 106:375–382PubMedCrossRefGoogle Scholar
  21. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659–1672PubMedCrossRefGoogle Scholar
  22. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Davidson NE Jr, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N (2005) Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353:1673–1684PubMedCrossRefGoogle Scholar
  23. Schlemmer BO, Sorensen BS, Overgaard J, Olsen KE, Gjerdrum LM, Nexo E (2004) Quantitative PCR–new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization. Scand J Clin Lab Invest 64:511–522PubMedCrossRefGoogle Scholar
  24. Schwarzenbach H, Muller V, Milde-Langosch K, Steinbach B, Pantel K (2011) Evaluation of cell-free tumour DNA and RNA in patients with breast cancer and benign breast disease. Mol BioSyst 7:2848–2854PubMedCrossRefGoogle Scholar
  25. Shaw JA, Smith BM, Walsh T, Johnson S, Primrose L, Slade MJ, Walker RA, Coombes RC (2000) Microsatellite alterations plasma DNA of primary breast cancer patients. Clin Cancer Res 6:1119–1124PubMedGoogle Scholar
  26. Shaw JA, Page K, Blighe K, Hava N, Guttery D, Ward B, Brown J, Ruangpratheep C, Stebbing J, Payne R, Palmieri C, Cleator S, Walker RA, Coombes RC (2012) Genomic analysis of circulating cell-free DNA infers breast cancer dormancy. Genome Res 22:220–231PubMedCrossRefGoogle Scholar
  27. Slamon DJ (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177–182PubMedCrossRefGoogle Scholar
  28. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792PubMedCrossRefGoogle Scholar
  29. Sorensen BS, Mortensen LS, Andersen J, Nexo E (2010) Circulating HER2 DNA after trastuzumab treatment predicts survival and response in breast cancer. Anticancer Res 30:2463–2468PubMedGoogle Scholar
  30. Steinman CR (1975) Free DNA in serum and plasma from normal adults. J Clin Invest 56:512–515PubMedCrossRefGoogle Scholar
  31. Swystun LL, Mukherjee S, Liaw PC (2011) Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus. J Thromb Haemost 9:2313–2321PubMedCrossRefGoogle Scholar
  32. Tse C, Brault D, Gligorov J, Antoine M, Neumann R, Lotz JP, Capeau J (2005) Evaluation of the quantitative analytical methods real-time PCR for HER-2 gene quantification and ELISA of serum HER-2 protein and comparison with fluorescence in situ hybridization and immunohistochemistry for determining HER-2 status in breast cancer patients. Clin Chem 51:1093–1101PubMedCrossRefGoogle Scholar
  33. Untch M, Fasching PA, Konecny GE, Hasmuller S, Lebeau A, Kreienberg R, Camara O, Muller V, du Bois A, Kuhn T, Stickeler E, Harbeck N, Hoss C, Kahlert S, Beck T, Fett W, Mehta KM, von Minckwitz G, Loibl S (2011) Pathologic complete response after neoadjuvant chemotherapy plus trastuzumab predicts favorable survival in human epidermal growth factor receptor 2-overexpressing breast cancer: results from the TECHNO trial of the AGO and GBG study groups. J Clin Oncol 29:3351–3357PubMedCrossRefGoogle Scholar
  34. von Minckwitz G, Loibl S, Maisch A, Untch M (2011) Lessons from the neoadjuvant setting on how best to choose adjuvant therapies. Breast 20(Suppl 3):S142–S145CrossRefGoogle Scholar
  35. Weigel MT, Dowsett M (2010) Current and emerging biomarkers in breast cancer: prognosis and prediction. Endocr Relat Cancer 17:R245–R262PubMedCrossRefGoogle Scholar
  36. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, Wheeler TM, Hayes DF (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25:118–145PubMedCrossRefGoogle Scholar
  37. Zanetti-Dallenbach R, Wight E, Fan AX, Lapaire O, Hahn S, Holzgreve W, Zhong XY (2008) Positive correlation of cell-free DNA in plasma/serum in patients with malignant and benign breast disease. Anticancer Res 28:921–925PubMedGoogle Scholar
  38. Zhong XY, Ladewig A, Schmid S, Wight E, Hahn S, Holzgreve W (2007) Elevated level of cell-free plasma DNA is associated with breast cancer. Arch Gynecol Obstet 276:327–331PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Troels Bechmann
    • 1
    • 2
    Email author
  • Rikke Fredslund Andersen
    • 3
  • Niels Pallisgaard
    • 3
  • Jonna Skov Madsen
    • 1
    • 3
  • Else Maae
    • 2
  • Erik Hugger Jakobsen
    • 2
  • Anne Marie Bak Jylling
    • 4
  • Karina Dahl Steffensen
    • 5
  • Anders Jakobsen
    • 1
    • 2
  1. 1.The Faculty of Health SciencesUniversity of Southern DenmarkOdenseDenmark
  2. 2.Department of OncologyVejle HospitalVejleDenmark
  3. 3.Department of BiochemistryVejle HospitalVejleDenmark
  4. 4.Department of PathologyOdense University HospitalOdenseDenmark
  5. 5.Department of OncologyOdense University HospitalOdenseDenmark

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