Login to MyKarger

New to MyKarger? Click here to sign up.



Login with Facebook

Forgot your password?

Authors, Editors, Reviewers

For Manuscript Submission, Check or Review Login please go to Submission Websites List.

Submission Websites List

Institutional Login
(Shibboleth or Open Athens)

For the academic login, please select your country in the dropdown list. You will be redirected to verify your credentials.

Original Article

Free Access

Real-Time qPCR-Based Detection of Circulating Tumor Cells from Blood Samples of Adjuvant Breast Cancer Patients: A Preliminary Study

Andergassen U. · Zebisch M. · Kölbl A.C. · König A. · Heublein S. · Schröder L. · Hutter S. · Friese K. · Jeschke U.

Author affiliations

Department of Obstetrics and Gynecology, Ludwig Maximilians University, Munich, Germany

Corresponding Author

Prof. Dr. Udo Jeschke

Department of Obstetrics and Gynecology

Ludwig Maximilians University of Munich

Maistrasse 11, 80337 Munich, Germany

udo.jeschke@med.uni-muenchen.de

Related Articles for ""

Breast Care 2016;11:194-198

Do you have an account?

Login Information





Contact Information











I have read the Karger Terms and Conditions and agree.



Summary

Background: Circulating tumor cells (CTCs) are cells that detach from a primary tumor, circulate through the blood stream and lymphatic vessels, and are considered to be the main reason for remote metastasis. Due to their origin, tumor cells have different gene expression levels than the surrounding blood cells. Therefore, they might be detectable in blood samples from breast cancer patients by real-time quantitative polymerase chain reaction (RT-qPCR). Materials and Methods: Blood samples of healthy donors and adjuvant breast cancer patients were withdrawn and the cell fraction containing white blood cells and tumor cells was enriched by density gradient centrifugation. RNA was isolated and reverse transcribed to cDNA, which was then used in TaqMan real-time PCR against cytokeratin (CK)8, CK18 and CK19. 18S and GAPDH were used as controls. Results: All 3 CKs were, on average, found to be significantly higher expressed in adjuvant breast cancer samples compared to negative controls, probably due to the presence of CTCs. Unfortunately, gene expression levels could not be correlated to tumor characteristics. Conclusions: RT-qPCR could make up a new approach for the detection of CTCs from blood samples of breast cancer patients, but a correlation of the PCR data to gold standard methods in CTC detection would help to further improve the informative value of the qPCR results.

© 2016 S. Karger GmbH, Freiburg


References

  1. Mocellin S, Keilholz U, Rossi CR, Nitti D: Circulating tumor cells: the ‘leukemic phase' of solid cancers. Trends Mol Med 2006;12:130-139.
  2. Pantel K, Brakenhoff RH: Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448-456.
  3. Ring A, Smith IE, Dowsett M: Circulating tumour cells in breast cancer. Lancet Oncol 2004;5:79-88.
  4. Smerage JB, Hayes DF: The measurement and therapeutic implications of circulating tumour cells in breast cancer. Br J Cancer 2006;94:8-12.
  5. Molloy TJ, Bosma AJ, van't Veer LJ: Towards an optimized platform for the detection, enrichment, and semi-quantitation of circulating tumor cells. Breast Cancer Res Treat 2008;112:297-307.
  6. Molloy TJ, Devriese LA, Helgason HH, Bosma AJ, Hauptmann M, Voest EE, Schellens JH, van't Veer LJ: A multimarker qPCR-based platform for the detection of circulating tumour cells in patients with early-stage breast cancer. Br J Cancer 2011;104:1913-1919.
  7. Xenidis N, Ignatiadis M, Apostolaki S, Perraki M, Kalbakis K, Agelaki S, Stathopoulos EN, Chlouverakis G, Lianidou E, Kakolyris S, Georgoulias V, Mavroudis D: Cytokeratin-19 mRNA-positive circulating tumor cells after adjuvant chemotherapy in patients with early breast cancer. J Clin Oncol 2009;27:2177-2184.
  8. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S, Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G: Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525-533.
  9. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G, Diel IJ, Gerber B, Gebauer G, Pierga JY, Marth C, Oruzio D, Wiedswang G, Solomayer EF, Kundt G, Strobl B, Fehm T, Wong GY, Bliss J, Vincent-Salomon A, Pantel K: A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353:793-802.
  10. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781-791.
  11. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, Somerfield MR, Hayes DF, Bast RC Jr: American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol 2007;25:5287-5312.
  12. Van der Auwera I, Peeters D, Benoy IH, Elst HJ, Van Laere SJ, Prove A, Maes H, Huget P, van Dam P, Vermeulen PB, Dirix LY: Circulating tumour cell detection: a direct comparison between the CellSearch system, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer. Br J Cancer 2010;102:276-284.
  13. Sieuwerts AM, Kraan J, Bolt-de Vries J, van der Spoel P, Mostert B, Martens JW, Gratama JW, Sleijfer S, Foekens JA: Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR. Breast Cancer Res Treat 2009;118:455-468.
  14. Sieuwerts AM, Mostert B, Bolt-de Vries J, Peeters D, de Jongh FE, Stouthard JM, Dirix LY, van Dam PA, Van Galen A, de Weerd V, Kraan J, van der Spoel P, Ramirez-Moreno R, van Deurzen CH, Smid M, Yu JX, Jiang J, Wang Y, Gratama JW, Sleijfer S, Foekens JA, Martens JW: mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients. Clin Cancer Res 2011;17:3600-3618.
  15. Stathopoulou A, Gizi A, Perraki M, Apostolaki S, Malamos N, Mavroudis D, Georgoulias V, Lianidou ES: Real-time quantification of CK-19 mRNA-positive cells in peripheral blood of breast cancer patients using the LightCycler system. Clin Cancer Res 2003;9:5145-5151.
    External Resources
  16. Androulakis N, Agelaki S, Perraki M, Apostolaki S, Bozionelou V, Pallis A, Kalbakis K, Xyrafas A, Mavroudis D, Georgoulias V: Clinical relevance of circulating CK-19 mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer. Br J Cancer 2012;106:1917-1925.
  17. Daskalaki A, Agelaki S, Perraki M, Apostolaki S, Xenidis N, Stathopoulos E, Kontopodis E, Hatzidaki D, Mavroudis D, Georgoulias V: Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer. Br J Cancer 2009;101:589-597.
  18. Ignatiadis M, Kallergi G, Ntoulia M, Perraki M, Apostolaki S, Kafousi M, Chlouverakis G, Stathopoulos E, Lianidou E, Georgoulias V, Mavroudis D: Prognostic value of the molecular detection of circulating tumor cells using a multimarker reverse transcription-PCR assay for cytokeratin 19, mammaglobin A, and Her2 in early breast cancer. Clin Cancer Res 2008;14:2593-2600.
  19. Wang HY, Ahn S, Kim S, Park S, Park S, Han H, Sohn JH, Kim S, Lee H: Detection of circulating tumor cells in patients with breast cancer using the quantitative RT-PCR assay for monitoring of therapy efficacy. Exp Mol Pathol 2014;97:445-452.
  20. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstappen LW: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 2004;10:6897-6904.
  21. Andergassen U, Kolbl AC, Hutter S, Friese K, Jeschke U: Detection of circulating tumour cells from blood of breast cancer patients via RT-qPCR. Cancers 2013;5:1212-1220.
  22. Zebisch M, Kolbl AC, Schindlbeck C, Neugebauer J, Heublein S, Ilmer M, Rack B, Friese K, Jeschke U, Andergassen U: Quantification of breast cancer cells in peripheral blood samples by real-time RT-PCR. Anticancer Res 2012;32:5387-5391.
    External Resources
  23. Zebisch M, Kolbl AC, Andergassen U, Hutter S, Neugebauer J, Engelstadter V, Gunthner-Biller M, Jeschke U, Friese K, Rack B: Detection of circulating tumour cells on mRNA levels with established breast cancer cell lines. Biomed Rep 2013;1:231-234.
  24. Kurec AS, Baltrucki L, Mason DY, Davey FR: Use of the APAAP method in the classification and diagnosis of hematologic disorders. Clin Lab Med 1988;8:223-236.
    External Resources
  25. Noack F, Schmitt M, Bauer J, Helmecke D, Kruger W, Thorban S, Sandherr M, Kuhn W, Graeff H, Harbeck N: A new approach to phenotyping disseminated tumor cells: methodological advances and clinical implications. Int J Biol Markers 2000;15:100-104.
    External Resources
  26. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408.
  27. Mostert B, Sleijfer S, Foekens JA, Gratama JW: Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer. Cancer Treat Rev 2009;35:463-474.
  28. Bleiweiss IJ, Nagi CS, Jaffer S: Axillary sentinel lymph nodes can be falsely positive due to iatrogenic displacement and transport of benign epithelial cells in patients with breast carcinoma. J Clin Oncol 2006;24:2013-2018.
  29. Diaz NM, Vrcel V, Centeno BA, Muro-Cacho C: Modes of benign mechanical transport of breast epithelial cells to axillary lymph nodes. Adv Anat Pathol 2005;12:7-9.
  30. Winkens T, Pachmann K, Freesmeyer M: Circulating epithelial cells in patients with thyroid carcinoma. Can they be identified in the blood? Nuklearmedizin 2013;52:7-13.
  31. Pantel K, Deneve E, Nocca D, Coffy A, Vendrell JP, Maudelonde T, Riethdorf S, Alix-Panabieres C: Circulating epithelial cells in patients with benign colon diseases. Clin Chem 2012;58:936-940.
  32. Rhim AD, Thege FI, Santana SM, Lannin TB, Saha TN, Tsai S, Maggs LR, Kochman ML, Ginsberg GG, Lieb JG, Chandrasekhara V, Drebin JA, Ahmad N, Yang YX, Kirby BJ, Stanger BZ: Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions. Gastroenterology 2014;146:647-651.
  33. Hofman VJ, Ilie MI, Bonnetaud C, Selva E, Long E, Molina T, Vignaud JM, Flejou JF, Lantuejoul S, Piaton E, Butori C, Mourad N, Poudenx M, Bahadoran P, Sibon S, Guevara N, Santini J, Venissac N, Mouroux J, Vielh P, Hofman PM: Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls. Am J Clin Pathol 2011;135:146-156.
  34. Andergassen U, Hofmann S, Kolbl AC, Schindlbeck C, Neugebauer J, Hutter S, Engelstadter V, Ilmer M, Friese K, Jeschke U: Detection of tumor cell-specific mRNA in the peripheral blood of patients with breast cancer - evaluation of several markers with real-time reverse transcription-PCR. Int J Mol Sci 2013;14:1093-1104.
  35. Guo M, Li X, Zhang S, Song H, Zhang W, Shang X, Zheng Y, Jiang H, Lv Q, Jiang Y, Hao H: Real-time quantitative RT-PCR detection of circulating tumor cells from breast cancer patients. Int J Oncol 2015;46:281-289.
  36. Smirnov DA, Zweitzig DR, Foulk BW, Miller MC, Doyle GV, Pienta KJ, Meropol NJ, Weiner LM, Cohen SJ, Moreno JG, Connelly MC, Terstappen LW, O'Hara SM: Global gene expression profiling of circulating tumor cells. Cancer Res 2005;65:4993-4997.
  37. Hayes DF, Walker TM, Singh B, Vitetta ES, Uhr JW, Gross S, Rao C, Doyle GV, Terstappen LW: Monitoring expression of Her-2 on circulating epithelial cells in patients with advanced breast cancer. Int J Oncol 2002;21:1111-1117.
  38. Chang HJ, Yang MJ, Yang YH, Hou MF, Hsueh EJ, Lin SR: MMP13 is potentially a new tumor marker for breast cancer diagnosis. Oncol Rep 2009;22:1119-1127.
  39. Nikseresht M, Seghatoleslam A, Monabati A, Talei A, Ghalati FB, Owji AA: Overexpression of the novel human gene, UBE2Q2, in breast cancer. Cancer Genet Cytogenet 2010;197:101-106.
  40. Fabre-Lafay S, Garrido-Urbani S, Reymond N, Goncalves A, Dubreuil P, Lopez M: Nectin-4, a new serological breast cancer marker, is a substrate for tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM-17. J Biol Chem 2005;280:19543-19550.
  41. Joosse SA, Hannemann J, Spotter J, Bauche A, Andreas A, Muller V, Pantel K: Changes in keratin expression during metastatic progression of breast cancer: impact on the detection of circulating tumor cells. Clin Cancer Res 2012;18:993-1003.
  42. Joosse SA, Pantel K: Biologic challenges in the detection of circulating tumor cells. Cancer Res 2013;73:8-11.

Article / Publication Details

First-Page Preview
Abstract of Original Article

Published online: June 15, 2016
Issue release date: June 2016

Number of Print Pages: 5
Number of Figures: 2
Number of Tables: 2

ISSN: 1661-3791 (Print)
eISSN: 1661-3805 (Online)

For additional information: https://www.karger.com/BRC


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.