Strahlentherapie

und Onkologie Original Article

Comparison of the Micronucleus and Chromosome

Aberration Techniques for the Documentation
of Cytogenetic Damage in Radiochemotherapy-Treated
Patients with Rectal Cancer
Hendrik Andreas Wolff*1, Steffen Hennies*1, Markus Karl Alfred Herrmann1, Margret Rave-Fränk1,
David Eickelmann1, Patricia Virsik2, Klaus Jung3, Markus Schirmer4, Michael Ghadimi5,
Clemens Friedrich Hess1, Robert Michael Hermann1,6, Hans Christiansen1

Purpose: The goal of the interdisciplinary Clinical Research Unit KFO179 (Biological Basis of Individual Tumor Response in Pa-
tients 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 lym-
phocytes. 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 increas-
ing 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

Strahlenther Onkol 2011;187:52–8

DOI 10.1007/s00066-010-2163-9

Vergleich von Mikronukleus- und Chromosomenaberrationstechnik für die Dokumentation zytogenetischer Schäden
bei neoadjuvant radiochemotherapierten Patienten mit Rektumkarzinom
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 Radiochemo-
therapie herauszuarbeiten, die im weiteren Verlauf der Arbeit der Forschergruppe Anwendung finden soll. Wir verglichen dabei
den Mikronukleustest (MN) mit der Chromosomenaberrationsanalyse (CAA).

1
Department of Radiotherapy and Radiooncology, University Medicine Göttingen, Göttingen, Germany,
2
Department of Hygiene and Environmental Medicine, University Medicine Göttingen, Göttingen, Germany,
3
Department of Medical Statistics, University Medicine Göttingen, Göttingen, Germany,
4
Department of Clinical Pharmacology, University Medicine Göttingen, Göttingen, Germany,
5
Department of Surgery, University Medicine Göttingen, Göttingen, Germany,
6
Department of Radiotherapy and Radiooncology, Ärztehaus an der Ammerlandklinik, Westerstede, Germany.
*H. A. Wolff and S. Hennies contributed equally to this work

Received: April 19, 2010; accepted: September 16, 2010

Published Online: December 23, 2010

52 Strahlenther Onkol 2011 · No. 1 © Urban & Vogel

Wolf HA, et al. Radiation-Induced Cytogenetic Damage in Rectal Cancer Patients

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 applizier-
ten 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-/Toxizi-
tätsscores 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.

Schlüsselworte: Rektumkarzinom · Neoadjuvante Radiochemotherapie · Mikronuklei · Chromosomaberrationstechnik ·

Lymphozyten

Introduction Patients and Methods

Neoadjuvant radiochemotherapy followed by surgery is the
standard approach for treatment of locally advanced rectal
cancer [21, 24, 30, 40, 43]. The interdisciplinary Clinical Re-
search Unit KFO 179 (Biological Basis of Individual Tumor
Response in Patients with Rectal Cancer) of the DFG (Ger-
man Research Foundation) aims to enhance the understand-
ing of the biological basis of tumor response and to establish
predictors of response and of treatment toxicity. The ultimate
goal is to develop an individual Response and Toxicity Score.
To document treatment response and toxicity, biological
and molecular parameters were collected in addition to clini-
cal data. Radio- (RTX) and chemotherapy (CTX) cause DNA
damage, which can be detected in whole blood lymphocytes
using the chromosome aberration (CA) technique (CAT) and
by the cytokinesis-block micronucleus (CBMN) assay.
Concerning the CAT, a dose-dependent increase in the
yield of CA frequency has been shown in RTX of patients at
different sites [2, 8, 26, 27]. It is a sensitive method for dose
estimation but requires a very detailed analysis, which is very
laborious and complex. A comparable increase in frequencies
of micronuclei during in vivo radiation [6, 11, 17, 38] has also
been described. It has been shown that the CBMN assay is
simple, reliable, and sensitive, resulting from the statistical
power afforded by a high scoring rate achievable in less time
[9, 12–14]. However, there is only little information about
the equivalence of the two methods, when lymphocytes are
exposed to DNA-damaging agents during clinical radioche-
motherapy. The aim of the present study was to compare the
results of the CAT and the CBMN assay in human lympho-
cytes of rectal cancer patients treated within the randomized
clinical trial CAO/ARO/AIO-04 (Chirurgische Arbeitsge-
meinschaft für Onkologie (CAO)/Arbeitsgemeinschaft Ra-
diologischer Onkologie (ARO)/Arbeitsgemeinschaft Inter-
nistische Onkologie (AIO)-04) of the German Rectal Cancer
Study Group.
Patients
From November 2007 to April 2008, 22 patients with locally
advanced rectal cancer were included in the study. All patients
were participating in the CAO/ARO/AIO-04 trial of the Ger-
man Rectal Cancer Study Group. In that study, patients with
locally advanced rectal cancer (UICC stage II/III) were treat-
ed in a neoadjuvant setting with radiotherapy (50.4 Gy) either
with standard chemotherapy with 5-fluorouracil (Arm A) or
with an experimental chemotherapy regimen using 5-FU and
oxaliplatin (Arm B). A summary of patient characteristics are
shown in Table 1.
Methods
Cell Cultures
To display cytogenetic damage, heparinized peripheral blood
samples were obtained from untreated patients before thera-
py (0 Gy) and during therapy after 21.6 Gy (12 fractions) and
50.4 Gy (28 fractions), respectively.
Chromosome Aberrations
Whole blood lymphocytes (6 ml per culture) were cultured
in 54 ml Roswell Park Memorial Institute medium (RPMI;
pH 7.2) containing 10% fetal calf serum, 100 µl/ml PHA, and
antibiotics (104 IU/ml penicillin and 10 mg/ml streptomycin
(GIBCO, Auckland, New Zealand)) in 5% CO2 atmosphere
at 37 °C. The culture times were 48 hours/50 hours, respec-
tively, taking into account individual variation. Colcemid
(20 µl/ml medium; GIBCO, Auckland, New Zealand) was
added before the last 2 hours of culturing. Metaphase cells
were prepared according to the standard method (hypotonic
0.56 M KCl; Merck, Darmstadt, Germany) treatment fol-
lowed by fixation in methanol (Merck, Darmstadt, Germany)
plus glacial acetic acid (3:1; Merck, Darmstadt, Germany) and
stored at 4 °C. The optimal culture time for cell preparations
containing not more than 6% of cells in their second postirra-
diation mitosi, was chosen according to the results of forego-
ing evaluation based upon fluorochrome-photolysis-Giemsa
(FPG) staining. This Giemsa/Hoechst-33258 staining method

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