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Cytogenetic features of leukaemias diagnosed in residentsof areas contaminated after the Chernobyl nuclear accident
E.V. Domrachevaa,*, E.A. Aseevaa, T.N. Obukhovaa, Y.N. Kobzeva,Y.V. Olshanskayaa, L.V. D'achenkoa, A.I. Udovichenkoa, A.V. Zakharovaa,
G.I. Milyutinab, V.V. Nechaic, A.I. Vorobiova
aKaryology Laboratory, National Research Center for Hematology, Russian Academy of Medical Sciences, Moscow, RussiabBryansk Regional Hospital, Bryansk, RussiacGomel Regional Hospital, Gomel, Byelorussia
Abstract
A comparison of chromosomal abnormalities in bone marrow leukaemic cells and of stable and unstable
aberrations in lymphocytes of patients with hematological malignancies who live in areas with or withoutcontamination by the Chernobyl nuclear accident has been made using FISH and G-banding. Healthy residents ofthese areas comprised the control group. No systematic cytogenetic di�erences of leukaemic cells between patients
from contaminated and uncontaminated areas were observed. Lymphocyte aberrations, however, were generallyhigher in all subjects from contaminated areas. Comparison has been made with speci®c cytogenetic features ofleukaemic cells and a high level of stable aberrations in lymphocytes of patients with secondary leukaemias that had
developed after chemo- and/or radio-therapy. 7 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
The problem of many people being exposed to radi-ation has become prominent during the last few dec-ades. Particularly in Russia, as also generally world-
wide, the number of radiation accidents has increasedand especially radiological events that have resulted inmany people being exposed. The problem of radiation-induced tumors, especially leukaemias, became urgent
after the Chernobyl accident. Several million residentsof contaminated areas of Russia, Ukraine, and Belor-ussia, and accident clean-up workers were exposed to
low LET radiation doses not exceeding 0.5 Gy. The
health implications, notably for stochastic diseases
such as leukaemia, are di�cult to assess and have led
to much debate and public concern.
There is overwhelming evidence that radiation is leu-
kaemogenic. This derives from studies on survivors of
the nuclear bombings in Japan and nuclear weapons
tests which led to a signi®cant increase of leukaemia
cases after the exposure to low doses of radiation (6±
30 cGy) (UNSCEAR, 1988). Occupational exposure of
radiologists (Ennov et al., 1989; Matanoski et al.,
1984), and therapeutic irradiation of patients with
spondylitis (Darby et al., 1985, 1988) or childhood thy-
mic hyperplasia (Ron et al., 1989) have also led to
demonstrable increases in leukaemia. Likewise, acci-
dental exposures such as at the Mayak accident in the
South Urals led to the same conclusions (Kossenko et
al., 1990). All these studies have demonstrated that ex-
Applied Radiation and Isotopes 52 (2000) 1171±1177
0969-8043/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S0969-8043(00 )00066-X
www.elsevier.com/locate/apradiso
* Corresponding author. Fax: +7-212-42-52.
E-mail address: dom@blood.ru (E.V. Domracheva).
posure to doses more than 0.5 Gy has a clear leukae-mogenic impact.
Already 12 years have passed since the Chernobylaccident, but still there is no really convincing evidenceof increased incidence of leukaemia in clean-up
workers and residents of contaminated areas who wereexposed to low doses of radiation.Analysis of samples from 48 clean-up workers with
leukaemia reported by the Obninsk National Radi-ation and Epidemiology Register suggested that 050%of the leukaemias might have been radiation induced
(Ivanov et al., 1996). In some regions of Ukraine, theleukaemia morbidity rate in contaminated areas hasbeen higher since the Chernobyl accident (Vigovskayaet al., 1994). By contrast, no signi®cant increase of leu-
kaemia in Bryansk, and Kaluga Regions as well as inother areas of Russia with contamination levels similarto the Ukranian study was noted (Osechinsky et al.,
1995). It should be emphasized that all previous studiesof leukaemia frequencies have been epidemiologicaland did not consider the cytology of every case.
A radiation aetiology could be postulated if there isgood documented evidence of radiation exposure. Im-mediately after the Chernobyl accident, an enhanced
level of unstable chromosomal aberrations served ascompelling evidence and now, years after the accident,such evidence could be a level of stable aberrations inlymphocytes (Stram et al., 1993). Certain cytogenetic
features of leukaemic cells could be an indicator of ir-radiation. These are hypodiploidy with monosomy ofchromosomes 5 or 7; deletions of long arms of these
chromosomes; trisomy 8; or less frequently, a translo-cation t(3;21) or t(1;7). Leukaemic cells with theseaberrations are characteristic for secondary leukaemias
developing after radiation exposure and/or chemother-apy.This study has tried to characterize bone marrow
leukaemic cells in patients living in radionuclide con-
taminated areas and to ascertain if any of the speci®ccytogenetic markers noted to be associated with sec-ondary leukaemias were especially evident. The fre-
quency of stable aberrations in lymphocytes of thesepatients was also established, so that this might serveas evidence of previous radiation exposure.
2. Materials and methods
2.1. Study population
The study was performed in 1989±1990 and in1996±1998. In the ®rst period, samples were investi-
gated from 313 healthy residents of grossly contami-nated (10±45 Ci/km2) areas of Gomel Region, andfrom 46 patients with hematological malignancies of T
able
1
Cytogenetic
methodsandsizesofgroupsstudied
Stable
aberrationsin
lymphocytes
Groupsstudied
G-bandingofbonemarrow
leukaem
iccells
FISH
G-band
Unstable
aberrationsin
lymphocytes
Residents
ofcontaminatedareas(Bryansk.obl.)
Acute
leukaem
ia48
24
10
CML
6
Healthy
37
22
Residents
ofnon-contaminatedareas
Acute
leukaem
ia151
18
11
CML
55
3
Healthy
33
18
70
Residents
ofcontaminatedareas(G
omel.obl.)
Acute
leukaem
ia38
CML
8
Healthy
313
Secondary
leukaem
ia12
12
4
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1171±11771172
comparable age range (18±73 years) who lived in the
same areas. In all these people, only the frequencies of
unstable aberrations were measured in the lymphocytes
as indicators of radiation exposure because of the
short period of elapsed time since the accident (Lucas
et al., 1992).
In 1996±1998, samples were studied from 54 resi-
dents diagnosed with leukaemia from the most con-
taminated areas of Bryansk Region, with 15±40 Ci/
km2. Their ages ranged from 23 to 67 years. Some of
these patients also had been Chernobyl clean-up
workers and some had been exposed to radiation after
the accident in the South Urals. Leukaemic marrow
cells were analysed by G-banding, and the frequency
of stable aberrations in their lymphocytes was
measured by FISH (24 persons) and G-banding (10
persons). Thirty-seven healthy residents of the same
contaminated areas (age range 24±53 years) comprised
the control group. They too were assayed for the fre-
quencies of stable aberrations in their lymphocytes
using FISH in all 37 persons and also with G-banding
in 22 of them.
A further study was made on 156 leukaemia living
in non-contaminated areas with the age range of 32±78
years, and from 33 healthy residents of the same areas
with no history of exposure to radiation (age range
24±56 years). Finally, data from these investigations
were compared with data from patients with secondary
leukaemias. All the groups studied are summarised in
Table 1.
2.2. Methods
G-banding was performed in a series of patients
from 1996±1998. Forty-eight hour standard cultures
with BrdU were employed for analysing stable and un-
stable aberrations in lymphocytes. The analysis of un-
stable aberrations was performed in 200±300 block
stained ®rst in vitro division metaphases. Stable aber-
rations in lymphocytes were studied by G-banding and
FISH (WCP1 1,2,4 Orange, Vysis, USA). A total of
50±100 metaphases were analysed by G-banding, and
438±2300 (650, median) metaphases by FISH. Aberra-
tions detected by G-banding were classi®ed according
to Ohtaki (1992) and those detected by FISH accord-
ing to Tucker et al. (1995). For extrapolating the aber-
ration frequencies in three pairs of chromosomes to
the whole genome, i.e. the genome equivalence, the
equation FG � Fp=2:05fp�1ÿ fp� was used. In this
equation, FG is the translocation frequency per whole
genome, Fp the translocation frequency observed in a
part of genome, and fp the genome portion for which
the DNA probes were used (Lucas et al., 1992).
3. Results and discussion
3.1. Comparative analysis of leukaemia types
Radiation-induced acute leukaemias in adults mostly
belong to M2, M3 and M5 types according to theFAB classi®cation and also to the so-called ``preleu-kaemias'' currently classi®ed as myelodysplastic syn-drome (MDS). The erythroid cell lineage is also
sensitive to radiation, and some investigators observean increase of acute myeloid-M6 leukaemias (AML)(Mole, 1986).
Fig. 1 shows data on the comparative frequencies ofleukaemia FAB-types in contaminated and non-con-taminated areas. The numbers of M2, M3 and M6 leu-
kaemia FAB-types diagnosed is approximately thesame in contaminated and non-contaminated areas(Fig. 1).
3.2. Cytogenetic features of leukaemic cells
The radiation-induced leukaemias are characterizedby speci®c cytogenetic aberrations (Le Beau et al.,
1986; Whang-Peng et al., 1988). Fig. 2 shows the pro-portion of such aberrations found in leukaemiapatients living in contaminated and non-contaminated
areas, as well as in patients with secondary leukaemias.Out of 48 acute leukaemia patients living in con-
taminated areas in the Bryansk oblast speci®c ``radi-
ation-induced'' cytogenetic changes were found in onlytwo cases (4.2%). In both patients it was trisomy 8. Inpatients from non-contaminated areas, such changes
were found in 8 out of 151 samples (5.3%), and inpatients with secondary leukaemias in 7 cases out of12 (58.3%). These results suggest that there is nodi�erence in cytogenetic features of leukaemic cells in
patients living in both, contaminated and ``clean''areas. By contrast at the same time, a much higherproportion of cytogenetic changes characteristic for
radiation-induced leukaemias were observed in thepatients with secondary leukaemias.It has been shown in many studies of nuclear acci-
dents that chronic myeloid leukaemia (CML) can becaused by irradiation. Some investigators pointed outthat such cases exhibit an exceptionally extended dur-ation of the disease; eight years or more. Samples from
the 6 CML Bryansk patients who lived in contami-nated areas were studied and a typical translocationt(9;22) was found in all of them, whilst the time
courses of the disease were unexceptional in ®ve ofthem.It is interesting to note that in samples from one
CML patient, this translocation was found not only inbone marrow but also in PHA-stimulated peripherallymphocytes. This prevented scoring the level of stable
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1171±1177 1173
aberrations in lymphocytes by G-banding or FISH.
The presence of t(9;22) in bone marrow and lympho-
cytes was con®rmed by performing FISH with a DNA
probe for t(9;22) (LSI2 bcr/abl Translocation probe,
Vysis, USA). This particular patient is still alive; the
disease is in chronic phase, and its duration is about 9
years. He was a clean-up worker at the site of an acci-
dent in South Urals, and EPR spectrometry analysis
Fig. 1. Frequencies of leukaemia FAB-types in contaminated and non-contaminated areas.
Fig. 2. Frequency (%) of secondary leukaemia speci®c chromosomal aberrations.
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1171±11771174
on a tooth showed that his absorbed dose was 35 cGy.These factors strongly suggest a radiation origin of this
particular CML case.
3.3. Unstable aberrations
In 1989±1990, the frequencies of unstable lympho-cyte aberrations were analysed in samples from 313
healthy people, 38 patients with leukaemias and MDS,and 8 CML patients, all living in the most contami-nated areas of Gomel Region. No karyotyping of the
leukaemic marrows was done for these subjects. Whencomparing by disease status, no obvious di�erences infrequencies of aberrant cells or that of dicentrics and
rings were observed in these people (Fig. 3). The per-cent frequency of aberrant lymphocytes in acute leu-kaemia patients from contaminated areas of Gomel
Region was 1.082 0.21; in MDS patients 0.902 0.24and in CML patients 0.820.31. After analyzing 48,531metaphases from healthy residents of the same terri-tories, with no history of radiation exposure, 1.23 20.11 aberrant cells per 100 metaphases were found.Thus, there were no signi®cant di�erences foundbetween the four groups.
The frequency of dicentrics plus rings per 100 cells(Fig. 3) was 0.182 0.02; 0.062 0.04; 0.142 0.06 foracute leukaemia patients (I), MDS patients (II) and
CML patients (III), respectively. The frequency ofdicentrics and rings in lymphocytes of healthy residentsof contaminated areas (IV) was 0.152 0.01. The con-trol group (V) comprised of 70 healthy residents of
non-contaminated areas with no history of radiationexposure. We analysed 19,950 metaphases fromsamples of these people, yielding 0.62 0.4% aberrant
cells with 0.0120.005% dicentrics plus rings.
3.4. Stable aberrations
The individual frequencies of stable aberrations cal-
culated per 100 metaphases (full genome) in all the
persons studied are presented in Fig. 4 and summar-
ised in Table 2. When comparing the frequencies of
stable aberrations in samples from patients with acute
leukaemia, MDS, and CML, who live in non-contami-
nated or contaminated areas (groups 2 and 4), with
frequencies of stable aberrations in samples from
healthy residents (groups 1 and 3), no di�erence was
found between healthy persons and leukaemia patients
living in the same areas. The frequency of stable aber-
rations established by FISH in patients with acute leu-
kaemia, MDS, and CML who live in non-
contaminated areas (group 2) varied from 0.2 to 6.7
per 100 metaphases (whole genome) (mean 1.6320.23)
and analysis by G-banding showed 0±2.0 stable aberra-
tions per 100 metaphases (mean 0.76 2 0.36). In
patients with acute leukaemias, MDS, and CML, who
live in contaminated areas (group 4), the frequencies
were as follows: by FISH 0.8±18.2 (mean 5.2122.10),
by G-banding 2.1±5.2 (mean 4.8121.30). It is very im-
portant to note that the translocation levels obtained
for patients with secondary leukaemias (group 5) are
signi®cantly higher than in all other groups studied.
The frequency of stable aberrations from health resi-
dents from non-contaminated areas (group 1) varied
from 0 to 1.9 (means 0.812 0.19 and 0.502 0.21 for
FISH and G-banding, respectively). In healthy resi-
dents of contaminated territories (group 3), the stable
aberration frequency observed by FISH varied from
0.6 to 15.3. This frequency, with means of 5.2721.22
and 4.992 0.97 per 100 cells measured by FISH and
G-banding, respectively, was signi®cantly higher than
in group 1 subjects; the healthy residents of non-con-
taminated territories. Group 1 comprises the control
subjects and the spontaneous level of translocations
found in them correlates well with frequencies pub-
lished by others; for example, 0.19 2 0.40 in new-
borns, and 2.392 0.24 in elderly subjects with a me-
dian 1.36 per 100 cells (Straume et al., 1992; Tucker,
Fig. 3. Frequency of unstable aberrations in lymphocytes of Gomel obl. residents (1989±1990).
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1171±1177 1175
1994; Tucker et al., 1994, 1995). The translocation fre-
quencies in healthy residents of contaminated terri-
tories (group 3) obtained in the present study are
elevated compared with the spontaneous incidence,
and are similar to those observed by Snigiryova et al.
(1997) in Chernobyl clean-up workers, namely 0.54±
7.40 per 100 cells (whole genome) using whole chromo-
some probes for 1, 2 and 12.
In one patient with AML-M2, the frequency of
translocations was very high at 18.2%. This person
was exposed to radiation after the Mayak accident in
1958, and then continued to live in one of the villages
along the grossly contaminated Techa River. It is not
feasible now to reconstruct his radiation dose but it
was surely high and so very likely to be responsible for
his leukaemia. Even so, some doubt must remain
because his leukaemia was not diagnosed until 40
years after the accident and did not exhibit any cytoge-
netic markers that have been speci®cally linked to ir-
radiation (the karyotype was 45, XY, -18).
It can be concluded that cases of acute leukaemia
and CML diagnosed in contaminated areas of
Bryansk were similar to the leukaemia cases diag-
nosed in non-contaminated areas with respect to
their FAB-type, and cytogenetic features of the leu-
kaemic cells. The level of stable aberrations in lym-
phocytes measured by FISH and G-banding was
signi®cantly higher in both healthy persons and leu-
kaemia patients living in contaminated areas, when
compared to the same groups of people living in
clean areas. In addition, no di�erence in frequencies
of stable aberrations was found between healthy
persons and leukaemia patients living in the same
areas. Ultimately, the level of stable aberrations in
lymphocytes observed both in healthy people and in
leukaemia patients depends only on the area in
which people live and not on their disease status.
Speci®c radiation-linked cytogenetic features as well
as elevated level of stable aberrations were found in
patients with secondary leukaemias. Monosomy and/or
deletion of chromosome 7 appeared to be the most fre-
quent ®ndings. However, very few of the primary leu-
Fig. 4. Frequency of state aberrations in leukaemia patients and healthy persons from contaminated and non-contaminated areas.
Table 2
Frequency of stable aberrations scored by FISH in investigated groups
Frequency of stable aberrations min value max value median mean
Healthy persons from non-contaminated areas 0 1.9 0.6 0.81
Persons with leukaemia from non-contaminated areas 0.2 6.7 1 1.63
Healthy persons from contaminated areas 0.6 15.3 4.8 5.27
Persons with leukaemia from contaminated areas 0.8 18.2 4.5 5.21
Persons with secondary leukaemia 8.2 16.8 12.55 12.80
E.V. Domracheva et al. / Applied Radiation and Isotopes 52 (2000) 1171±11771176
kaemia patients studied showed such characteristickaryotyping changes in their marrow cells.
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