Strahlentherapie und Onkologie

, Volume 187, Issue 1, pp 52–58 | Cite as

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

  • Hendrik Andreas Wolff*
  • Steffen Hennies
  • Markus Karl Alfred Herrmann
  • Margret Rave-Fränk
  • David Eickelmann
  • Patricia Virsik
  • Klaus Jung
  • Markus Schirmer
  • Michael Ghadimi
  • Clemens Friedrich Hess
  • Robert Michael Hermann
  • Hans Christiansen
Original Article

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.

Key Words

Rectal cancer Neoadjuvant radiochemotherapy Micronuclei Chromosome aberration technique Lymphocytes 

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

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.

Schlüsselwörter

Rektumkarzinom Neoadjuvante Radiochemotherapie Mikronuklei Chromosomaberrationstechnik Lymphozyten 

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References

  1. 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.CrossRefGoogle Scholar
  2. 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.CrossRefPubMedGoogle Scholar
  3. 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.PubMedGoogle Scholar
  4. 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.PubMedGoogle Scholar
  5. 5.
    Brunner E., Domhof S, Langer F. Nonparametric analysis of longitudinal data in factorial experiments. New York: Wiley 2002:187–210.Google Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. 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.CrossRefPubMedGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 9.
    Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc 2007;2:1084–104.CrossRefPubMedGoogle Scholar
  10. 10.
    Fenech M. The in vitro micronucleus technique. Mutat Res 2000;455:81–95.PubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 12.
    Fenech M, Morley A. Solutions to the kinetic problem in the micronucleus assay. Cytobios 1985;43:233–46.PubMedGoogle Scholar
  13. 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.PubMedGoogle Scholar
  14. 14.
    Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985;147:29–36.PubMedGoogle Scholar
  15. 15.
    Fernandes TS, Lloyd D, Amaral A. A comparison of different cytological stains for biological dosimetry. Int J Radiat Biol 2008;84:703–11.CrossRefPubMedGoogle Scholar
  16. 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.CrossRefPubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.CrossRefPubMedGoogle Scholar
  19. 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.PubMedGoogle Scholar
  20. 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.CrossRefPubMedGoogle Scholar
  21. 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.CrossRefPubMedGoogle Scholar
  22. 22.
    Kormos C, Koteles GJ. Comparison of cytogenetic tests for monitoring overexposures from ionizing radiation. Acta Biol Hung 1990;41:115–20.PubMedGoogle Scholar
  23. 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.PubMedGoogle Scholar
  24. 24.
    Marquardt F, Rodel F, Capalbo G, et al. Molecular targeted treatment and radiation therapy for rectal cancer. Strahlenther Onkol 2009;185:371–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Mateuca R, Lombaert N, Aka PV, et al. Chromosomal changes: induction, detection methods and applicability in human biomonitoring. Biochimie 2006;88:1515–31.CrossRefPubMedGoogle Scholar
  26. 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.CrossRefGoogle Scholar
  27. 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.PubMedGoogle Scholar
  28. 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.CrossRefPubMedGoogle Scholar
  29. 29.
    Parker WB, Cheng YC. Metabolism and mechanism of action of 5-fluorouracil. Pharmacol Ther 1990;48:381–95.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. 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.CrossRefPubMedGoogle Scholar
  32. 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.CrossRefPubMedGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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.CrossRefPubMedGoogle Scholar
  37. 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.CrossRefPubMedGoogle Scholar
  38. 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.CrossRefPubMedGoogle Scholar
  39. 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.CrossRefPubMedGoogle Scholar
  40. 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.CrossRefPubMedGoogle Scholar
  41. 41.
    Westlake WJ. Response to T.B.L. Kirkwood: bioequivalence testing—a need to rethink. Biometrics 1981;37:589–94.CrossRefGoogle Scholar
  42. 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.CrossRefPubMedGoogle Scholar
  43. 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.CrossRefPubMedGoogle Scholar
  44. 44.
    Woynarowski JM, Faivre S, Herzig MC, et al. Oxaliplatin-induced damage of cellular DNA. Mol Pharmacol 2000;58:920–7.PubMedGoogle Scholar
  45. 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.CrossRefPubMedGoogle Scholar
  46. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Urban & Vogel, Muenchen 2010

Authors and Affiliations

  • Hendrik Andreas Wolff*
    • 1
  • Steffen Hennies
    • 1
  • Markus Karl Alfred Herrmann
    • 1
  • Margret Rave-Fränk
    • 1
  • David Eickelmann
    • 1
  • Patricia Virsik
    • 2
  • Klaus Jung
    • 3
  • Markus Schirmer
    • 4
  • Michael Ghadimi
    • 5
  • Clemens Friedrich Hess
    • 1
  • Robert Michael Hermann
    • 1
    • 6
  • Hans Christiansen
    • 1
    • 7
  1. 1.Department of Radiotherapy and RadiooncologyUniversity Medicine GöttingenGöttingenGermany
  2. 2.Department of Hygiene and Environmental MedicineUniversity Medicine GöttingenGöttingenGermany
  3. 3.Department of Medical StatisticsUniversity Medicine GöttingenGöttingenGermany
  4. 4.Department of Clinical PharmacologyUniversity Medicine GöttingenGöttingenGermany
  5. 5.Department of SurgeryUniversity Medicine GöttingenGöttingenGermany
  6. 6.Department of Radiotherapy and RadiooncologyÄrztehaus an der AmmerlandklinikWesterstedeGermany
  7. 7.Department of Radiotherapy and RadiooncologyUniversity Medicine GoettingenGöttingenGermany

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