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Comparison of the Micronucleus and Chromosome Aberration Techniques for the Documentationof Cytogenetic Damage in Radiochemotherapy-Treated Patients with Rectal Cancer

Vergleich von Mikronukleus- und Chromosomenaberrationstechnik für die Dokumentation zytogenetischer Schäden bei neoadjuvant radiochemotherapierten Patienten mit Rektumkarzinom

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Abstract

Purpose:

The goal of the interdisciplinary Clinical Research Unit KFO179 (Biological Basis of Individual Tumor Response in Patients with Rectal Cancer) is to develop an individual Response and Toxicity Score for patients with locally advanced rectal cancer treated with neoadjuvant radiochemotherapy. The aim of the present study was to find a reliable and sensitive method with easy scoring criteria and high numbers of cell counts in a short period of time in order to analyze DNA damage in peripheral blood lymphocytes. Thus, the cytokinesis-block micronucleus (CBMN) assay and the chromosome aberration technique (CAT) were tested.

Materials and Methods:

Peripheral blood lymphocytes obtained from 22 patients with rectal cancer before (0 Gy), during (21.6 Gy), and after (50.4 Gy) radiochemotherapy were stimulated in vitro by phytohemagglutinin (PHA); the cultures were then processed for the CBMN assay and the CAT to compare the two methods.

Results:

A significant increase of chromosomal damage was observed in the course of radiochemotherapy parallel to increasing radiation doses, but independent of the chemotherapy applied. The equivalence of both methods was shown by Westlake’s equivalence test.

Conclusion:

The results show that the CBMN assay and the CAT are equivalent. For further investigations, we prefer the CBMN assay, because it is simpler through easy scoring criteria, allows high numbers of cell counts in less time, is reliable, sensitive, and has higher statistical power. In the future, we plan to integrate cytogenetic damage during radiochemotherapy into the planned Response and Toxicity Score within our interdisciplinary Clinical Research Unit.

Zusammenfassung

Ziel:

Ziel der interdisziplinären Klinischen Forschergruppe KFO179 (Biological Basis of Individual Tumor Response in Patients with Rectal Cancer) ist es, einen individuellen Response-/Toxizitätsscore für Patienten zu entwickeln, die bei diagnostiziertem lokal fortgeschrittenem Rektumkarzinom mit neoadjuvanter Radiochemotherapie behandelt werden. Ziel der vorliegenden Arbeit war, eine einfache und zuverlässige Methode zur Detektion des individuellen zytogenetischen Schadens durch die Radiochemotherapie herauszuarbeiten, die im weiteren Verlauf der Arbeit der Forschergruppe Anwendung finden soll. Wir verglichen dabei den Mikronukleustest (MN) mit der Chromosomenaberrationsanalyse (CAA).

Patienten und Methodik:

Periphere Blutlymphozyten von 22 Patienten wurden vor (0 Gy), während (21,6 Gy) und nach (50,4 Gy) Radiochemotherapie untersucht. Der zytogenetische Schaden wurde mittels MN und CAA analysiert und die Äquivalenz beider Methoden geprüft.

Ergebnisse:

Eine signifikante Zunahme chromosomaler Schädigungen durch die Bestrahlung in Abhängigkeit von der applizierten Dosis konnte bei beiden Techniken, unabhängig von der applizierten Chemotherapie, beobachtet werden. Die Gleichwertigkeit beider Methoden konnte durch den Äquivalenztest nach Westlake gezeigt werden.

Schlussfolgerung:

Es zeigte sich eine Äquivalenz der angewandten Methoden, was uns nun die Möglichkeit bietet, den MN gleichwertig gegenüber der CAA anzuwenden und für die geplanten Analysen bezüglich des individuellen Response-/Toxizitätsscores zu verwenden. Die Mikronukleustechnik ermöglicht durch leichtere Zählkriterien in kürzerer Zeit eine größere Anzahl von Zellen zu zählen, was zu einem valideren statistischen Endergebnis führt.

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References

  1. Almeida GM, Duarte TL, Steward WP, et al. Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay. DNA Repair (Amst) 2006;5:219–25.

    Article  CAS  Google Scholar 

  2. Antoine JL, Gerber GB, Leonard A, et al. Chromosome aberrations induced in patients treated with telecobalt therapy for mammary carcinoma. Radiation Research 1981;86:171–7.

    Article  CAS  PubMed  Google Scholar 

  3. Bahl A, Chander S, Julka PK, et al. Micronuclei evaluation of reduction in neoadjuvant chemotherapy related acute toxicity in locally advanced lung cancer: an indian experience. J Assoc Physicians India 2006;54:191–5.

    CAS  PubMed  Google Scholar 

  4. Bilban-Jakopin C, Bilban M. Genotoxic effects of radiotherapy and chemotherapy on circulating lymphocytes in patients with Hodgkin’s disease. Mutat Res 2001;497:81–8.

    CAS  PubMed  Google Scholar 

  5. Brunner E., Domhof S, Langer F. Nonparametric analysis of longitudinal data in factorial experiments. New York: Wiley 2002:187–210.

  6. Catena C, Conti D, Parasacchi P et al. Micronuclei in cytokinesis-blocked lymphocytes may predict patient response to radiotherapy. Int J Radiat Biol 1996;70:301–8.

    Article  CAS  PubMed  Google Scholar 

  7. Das B, Karuppasamy CV. Spontaneous frequency of micronuclei among the newborns from high level natural radiation areas of Kerala in the southwest coast of India. Int J Radiat Biol 2009;85:272–80.

    Article  CAS  PubMed  Google Scholar 

  8. Diener A, Stephan G, Vogl T, et al. The induction of chromosome aberrations during the course of radiation therapy for morbus Hodgkin. Radiat Res 1988;114:528–36.

    Article  CAS  PubMed  Google Scholar 

  9. Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc 2007;2:1084–104.

    Article  CAS  PubMed  Google Scholar 

  10. Fenech M. The in vitro micronucleus technique. Mutat Res 2000;455:81–95.

    CAS  PubMed  Google Scholar 

  11. Fenech M, Denham J, Francis W, et al. Micronuclei in cytokinesis-blocked lymphocytes of cancer patients following fractionated partial-body radiotherapy. Int J Radiat Biol 1990;57:373–83.

    Article  CAS  PubMed  Google Scholar 

  12. Fenech M, Morley A. Solutions to the kinetic problem in the micronucleus assay. Cytobios 1985;43:233–46.

    CAS  PubMed  Google Scholar 

  13. Fenech M, Morley AA. Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat Res 1986;161:193–8.

    CAS  PubMed  Google Scholar 

  14. Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985;147:29–36.

    CAS  PubMed  Google Scholar 

  15. Fernandes TS, Lloyd D, Amaral A. A comparison of different cytological stains for biological dosimetry. Int J Radiat Biol 2008;84:703–11.

    Article  CAS  PubMed  Google Scholar 

  16. Gamulin M, Kopjar N, Grgic, M et al. Genome damage in oropharyngeal cancer patients treated by radiotherapy. Croat Med J 2008;49:515–27.

    Article  PubMed  Google Scholar 

  17. Gantenberg HW, Wuttke K, Streffer C, et al. Micronuclei in human lymphocytes irradiated in vitro or in vivo. Radiat Res 1991;128:276–81.

    Article  CAS  PubMed  Google Scholar 

  18. Garaj-Vrhovac V, Fucic A, Horvat D. The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro. Mutat Res 1992;281:181–6.

    Article  CAS  PubMed  Google Scholar 

  19. Hatayoglu SE, Orta T. Relationship between radiation induced dicentric chromosome aberrations and micronucleus formation in human lymphocytes. J Exp Clin Cancer Res 2007;26:229–34.

    CAS  PubMed  Google Scholar 

  20. Hoskins J, Scott Butler J. Evidence for distinct DNA- and RNA-based mechanisms of 5-fluorouracil cytotoxicity in Saccharomyces cerevisiae. Yeast 2007;24:861–70.

    Article  CAS  PubMed  Google Scholar 

  21. Keilholz L, Mese M, Henneking K, et al. Effect of total mesorectal excision on the outcome of rectal cancer after standardized postoperative radiochemotherapy: do randomized studies translate into clinical routine? Strahlenther Onkol 2009;185:364–70.

    Article  PubMed  Google Scholar 

  22. Kormos C, Koteles GJ. Comparison of cytogenetic tests for monitoring overexposures from ionizing radiation. Acta Biol Hung 1990;41:115–20.

    CAS  PubMed  Google Scholar 

  23. Lee TK, O’Brien KF, Naves JL, et al. Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy. Mutat Res 2000;469:63–70.

    CAS  PubMed  Google Scholar 

  24. Marquardt F, Rodel F, Capalbo G, et al. Molecular targeted treatment and radiation therapy for rectal cancer. Strahlenther Onkol 2009;185:371–8.

    Article  PubMed  Google Scholar 

  25. Mateuca R, Lombaert N, Aka PV, et al. Chromosomal changes: induction, detection methods and applicability in human biomonitoring. Biochimie 2006;88:1515–31.

    Article  CAS  PubMed  Google Scholar 

  26. Matsubara S, Sasaki MS, Adachi T. Dose-response relationship of lymphocyte chromosome aberrations in locally irradiated persons. Journal of Radiation Research (Tokyo) 1974;15:189–96.

    Article  CAS  Google Scholar 

  27. Obe G, Matthiessen W, Gobel D. Chromosomal aberrations in the peripheral lymphocytes of cancer patients treated with high-energy electrons and bleomycin. Mutat Res 1981;81:133–41.

    CAS  PubMed  Google Scholar 

  28. Padjas A, Lesisz D, Lankoff A, et al. Cytogenetic damage in lymphocytes of patients undergoing therapy for small cell lung cancer and ovarian carcinoma. Toxicol Appl Pharmacol 2005;209:183–91.

    Article  CAS  PubMed  Google Scholar 

  29. Parker WB, Cheng YC. Metabolism and mechanism of action of 5-fluorouracil. Pharmacol Ther 1990;48:381–95.

    Article  CAS  PubMed  Google Scholar 

  30. Paskeviciute B, Bolling T, Brinkmann M, et al. Impact of (18)F-FDG-PET/CT on staging and irradiation of patients with locally advanced rectal cancer. Strahlenther Onkol 2009;185:260–5.

    Article  PubMed  Google Scholar 

  31. Pathak R, Sarma A, Sengupta B, et al. Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells. Int J Radiat Biol 2007;83:53–63.

    Article  CAS  PubMed  Google Scholar 

  32. Rigaud O, Guedeney G, Duranton I, et al. Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. I. Chromosome aberrations induced in vivo. Mutat Res 1990;242: 17–23.

    Article  CAS  PubMed  Google Scholar 

  33. Sala-Trepat M, Cole J, Green MH, et al. Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. III: Measurement of mutant frequency to 6-thioguanine resistance. Mutagenesis 1990;5:593–8.

    Article  CAS  PubMed  Google Scholar 

  34. Seoane A, Guerci A, Dulout F. Genetic instability induced by low doses of x-rays in hamster cells. Int J Radiat Biol 2007;83:81–7.

    Article  CAS  PubMed  Google Scholar 

  35. Singh S, Datta NR, Krishnani N, et al. Radiation therapy induced micronuclei in cervical cancer–does it have a predictive value for local disease control? Gynecol Oncol 2005;97:764–71.

    Article  PubMed  Google Scholar 

  36. Slonina D, Biesaga B, Urbanski K, et al. Comparison of chromosomal radiosensitivity of normal cells with and without HRS-like response and normal tissue reactions in patients with cervix cancer. Int J Radiat Biol 2008;84:421–8.

    Article  CAS  PubMed  Google Scholar 

  37. Stephan G, Schneider K, Panzer W, et al. Enhanced yield of chromosome aberrations after CT examinations in paediatric patients. Int J Radiat Biol 2007;83:281–7.

    Article  CAS  PubMed  Google Scholar 

  38. Thierens H, Vral A, Van Eijkeren M, et al. Micronucleus induction in peripheral blood lymphocytes of patients under radiotherapy treatment for cervical cancer or Hodgkin’s disease. Int J Radiat Biol 1995;67:529–39.

    Article  CAS  PubMed  Google Scholar 

  39. Thomas P, Umegaki K, Fenech M. Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay. Mutagenesis 2003;18:187–94.

    Article  CAS  PubMed  Google Scholar 

  40. Vorwerk H, Liersch T, Rothe H, et al. Gold markers for tumor localization and target volume delineation in radiotherapy for rectal cancer. Strahlenther Onkol 2009;185:127–33.

    Article  PubMed  Google Scholar 

  41. Westlake WJ. Response to T.B.L. Kirkwood: bioequivalence testing—a need to rethink. Biometrics 1981;37:589–94.

    Article  Google Scholar 

  42. Widel M, Jedrus S, Lukaszczyk B, et al. Radiation-induced micronucleus frequency in peripheral blood lymphocytes is correlated with normal tissue damage in patients with cervical carcinoma undergoing radiotherapy. Radiat Res 2003;159:713–21.

    Article  CAS  PubMed  Google Scholar 

  43. Wolff HA, Gaedcke J, Jung K, et al. High-grade acute organ toxicity during preoperative radiochemotherapy as positive predictor for complete histopathologic tumor regression in multimodal treatment of locally advanced rectal cancer. Strahlenther Onkol 2010;186:30–5.

    Article  PubMed  Google Scholar 

  44. Woynarowski JM, Faivre S, Herzig MC, et al. Oxaliplatin-induced damage of cellular DNA. Mol Pharmacol 2000;58:920–7.

    CAS  PubMed  Google Scholar 

  45. Wuttke K, Müller W-U, Streffer C. The sensitivity of the in vitro cytokinesisblock micronucleus assay in lymphocytes for different and combined radiation qualities. Strahlenther Onkol 1998;174:262–8.

    Article  CAS  PubMed  Google Scholar 

  46. Xuncla M, Barquinero JF, Caballin MR, et al. Cytogenetic damage induced by radiotherapy. Evaluation of protection by amifostine and analysis of chromosome aberrations persistence. Int J Radiat Biol 2008;84:243–51.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Radiotherapy and Radiooncology, University Medicine Göttingen, Göttingen, Germany

    Hendrik Andreas Wolff*, Steffen Hennies, Markus Karl Alfred Herrmann, Margret Rave-Fränk, David Eickelmann, Clemens Friedrich Hess, Robert Michael Hermann &  Hans Christiansen

  2. Department of Hygiene and Environmental Medicine, University Medicine Göttingen, Göttingen, Germany

    Patricia Virsik

  3. Department of Medical Statistics, University Medicine Göttingen, Göttingen, Germany

    Klaus Jung

  4. Department of Clinical Pharmacology, University Medicine Göttingen, Göttingen, Germany

    Markus Schirmer

  5. Department of Surgery, University Medicine Göttingen, Göttingen, Germany

    Michael Ghadimi

  6. Department of Radiotherapy and Radiooncology, Ärztehaus an der Ammerlandklinik, Westerstede, Germany

    Robert Michael Hermann

  7. Department of Radiotherapy and Radiooncology, University Medicine Goettingen, Robert-Koch-Strasse 40, 37075, Göttingen, Germany

    Hans Christiansen

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  1. Hendrik Andreas Wolff*
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  2. Steffen Hennies
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Correspondence to Hans Christiansen.

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*H. A. Wolff and S. Hennies contributed equally to this work

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Wolff*, H.A., Hennies, S., Herrmann, M.K.A. et al. Comparison of the Micronucleus and Chromosome Aberration Techniques for the Documentationof Cytogenetic Damage in Radiochemotherapy-Treated Patients with Rectal Cancer. Strahlenther Onkol 187, 52–58 (2011). https://doi.org/10.1007/s00066-010-2163-9

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  • DOI: https://doi.org/10.1007/s00066-010-2163-9

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