Download Normal Sister Chromatid Exchange Frequency

[CANCER RESEARCH 42. 2906-2908,
0008-5472/82/0042-OOOOS02.00
July 1982]
Normal Sister Chromatid Exchange Frequency in Long-Term Survivors
with Acute Leukemia1
Susumu Inoue2, Laura Brown, Yaddanapudi Ravindranath, and Mark J. Ottenbreit
Children s Hospital of Michigan [S. I., L. B., Y. R.¡,and Department
Michigan 48201
of Pediatrics,
ABSTRACT
We hypothesized that the sister chromatid exchanges assay
in acute leukemia long-term survivors may detect: (a) long-term
effects of combined chemo- and radiotherapy; and possibly (b)
those individuals with inherently deficient DMA repair. Accord
ingly, we determined the sister chromatid exchanges frequency
in 26 blood specimens from 24 acute leukemia long-term
survivors (patients) and 14 blood specimens from 13 control
subjects (controls). The patients consisted of 23 children with
acute lymphocytic and one child with acute myelocytic leuke
mia. The median length of chemotherapy was 5 years. Eighteen
of the 24 patients also received prophylactic fractional central
nervous system irradiation for the first 3 years of treatment,
and one patient received therapeutic irradiation to the central
nervous system. The median off-therapy period at the time of
study was 2.5 years with a range of 0 to 7.5 years.
The controls consisted of the parents of the patients and
laboratory personnel. A mean exchange score per cell was
established for each specimen (25 to 30 cells/specimen were
scored), and it ranged from 3.0 to 9.7 in the patients and from
3.0 to 11.5 in the controls. A mean ± S.D. calculated from
those means was 6.0 ±1.8 for the patients and 6.9 ±2.8 for
the controls. They were not significantly different. We conclude
that chemo- and radiotherapy produced no persistent DMA
alterations detectable by this method.
INTRODUCTION
Prognosis of ALL3 in children has improved markedly, and
more than 50% of the patients now achieve long uninterrupted
remission and are taken off chemotherapy (16). About 15% of
these children, however, relapse following the cessation of
therapy (16). The majority of these relapses is presumed to be
a relapse of the original leukemia. An alternative mechanism of
a relapse would be a new mutation or cancer transformation
after the original leukemic cells were completely irradicated.
That this could occur has been amply documented in the
literature (3, 4,13,19).
It could be hypothesized that some of
the relapses that occur after cessation of treatment may be in
reality due to the emergence of a new leukemic clone, as
opposed to the resurgence of the original leukemic cells. It has
become evident recently that patients treated with chemother
apy and/or radiotherapy for the primary cancer are at an
increased risk of developing a second cancer, including leu' Supported by Children's Leukemia Foundation of Michigan.
2 To whom requests for reprints should be addressed, at: Children's Hospital
of Michigan, 3901 Beaubien Boulevard, Detroit, Mich, 48201.
3 The abbreviations used are: ALL, acute lymphocytic leukemia; SCE, sister
chromatid exchange.
Received December 7. 1981; accepted March 30, 1982.
2906
Wayne State University School of Medicine [S. I., Y. R., M. J. O.], Detroit,
kemia, suggesting that these therapy modalities were directly
responsible for malignant mutation (3, 12, 18). In addition to
the exogenous factors, "proneness"
of the individual to de
velop cancer (or genetic factors of
considered. For example, so-called
syndrome characterized by increased
frequency, such as Fanconi's anemia
the host) must also be
chromosomal instability
chromosome breakage
or Bloom's syndrome, is
an established heritable condition with an increased risk of
cancer.
Recent investigations of another chromosomal phenomenon,
SCE, showed that the SCE assay would be a simple and
sensitive assay system to determine mutagenicity of various
carcinogens (extrinsic factors) (15, 20) and perhaps to detect
DNA repair deficiency (one of the host factors) (20). If some of
the relapses that occur after prolonged uninterrupted remission
are due to a new mutation event, this could be due to: (a)
prolonged exposure to chemotherapeutic agents and/or irra
diation; (b) inherent susceptibility of the host to develop cancer
repeatedly; or (c) a combination of both. We hypothesized that
these factors, singly or in combination, may be reflected as an
increased SCE frequency.
We have conducted a prospective study to test this hypoth
esis, and this paper describes our preliminary findings of this
ongoing study.
MATERIALS
AND METHODS
Study Population. The patient group consisted of 24 children with
acute leukemia (all but one with ALL) who had been in remission for
5 years or longer and were no longer receiving any treatment at the
time of study. These 24 patients received chemotherapy for 3 to 7.25
years with a median of 5 years. In addition, 19 of the 24 patients
received prophylactic fractional central nervous system irradiation to
the skull and spine (100 rads to each) every 10 weeks for the first 3
years (15 courses). Three of the patients had received 2 courses of
chemotherapy because they relapsed while off treatment. At the time
of the study, the off-therapy period ranged from 0.25 to 7.25 years
with a median of 2.5 years. In 23 of the 24 patients, the chemotherapy
consisted of a cyclic treatment of vincristine, 6-mercaptopurine,
and
prednisone alternated with p.o. mpthotrexate (Protocols I and II) (6).
One patient received only p.o. 6-mercaptopurine,
methotrexate, and
prednisone (composite cyclic therapy) (6). Details of the patients are
shown in Table 1. As of this writing, none of the patients relapsed. The
control group consisted of 13 individuals (6 parents of the patients and
7 laboratory personnel).
Method. Ten ml of heparinized blood were drawn, and the RBC were
sedimented. The buffy coat cells were washed twice in a-medium and
cultured in the dark in a-medium without thymidine with 20% fetal calf
serum and with 25 fig 5-bromodeoxyuridine
per ml of media for 3 days.
Harvesting of the cells and preparation of the slides were made
according to the method described previously (11). The slides were
then exposed to fluorescent light for 15 min that was generated by an
Osram HBO 220-watt high-pressure mercury lamp that was fitted to a
CANCER
RESEARCH
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VOL. 42
SCE in Leukemia Survivors
Table 1
groupPatient12«3456a7891011121314a15161718192021222324DiagnosisALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALLALL
Patient
systemirradia
oftreatment5yr8
tionYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesNoNoNoNoNoOff-therapyperiod03
mos.5yr4
yr 6
mos.9
mos.1yr1
mos.5yr9yr5yr5yr3yr5yrSyr5yr4yr9yr5yr5yr5yr5yr5yr5
yr 6
yr1
mos.1
yr 6
mos.1
yr 8
mos.1
yr 9
mos.1
yr 9
mos.2yr2
yr 10
mos.2
yr 6
mos.2
yr 6
mos.2
yr 6
mos.3
yr 8
mos.3
yr 2
mos.3
yr 3
mos.3
yr 6
mos.4yr4
yr 6
mos.SyrSyr5
yr 10
mos.Syr7
yr 6
mos.7yr7yrCentralnervous
yr 3
mos.6
yr 6
mos.7
yr 8
yr 6 mos.Range0-82-90-162-123-181-92-122-182-114-140-232-150-110-91-123-142-102-142-111-13
±1.8°
a In Cases 6 and 14, a bone marrow relapse; and in Case 2, a meningeal elapse occurred off therapy. A
second course of chemotherapy and prophylactic irradiation were given in the former 2 and therapeutic
irradiation were given in the third. Off-therapy period in these patients indicates the time after cessation of
the second course.
6 AML, acute myelocytic leukemia.
c Mean ±S.D.
Zeiss microscope and filtered through a Zeiss BG12 filter. The slides
were heated to 60° in 2-fold standard saline citrate (0.15 M sodium
Table 2
Control group
chloride:0.015
M sodium citrate, pH 7.4), for 2 hr and stained with
Giemsa. At least 25 cells with a maximum of 30 cells were analyzed for
the SCE frequency.
SCE
Range
Mean
Nonsmokers12345678910Smokers111213Control0-110-80-142-80-60-63-162-122-122-135-284-203
RESULTS
The results are shown in Table 2. In summary, for the patient
group, the mean SCE frequency for each specimen ranged
from 3.0 to 9.7/cell. The mean ± S.D. of these individual
means was 6.0 ±1.8. For the control group, the mean ranged
from 3.0 to 11.5/cell. The mean ±S.D. for the control group
was 6.9 ± 2.8. When the control group was divided into
smokers (more than 1 pack/day) and nonsmokers, mean ±
S.D. for the former was 10.7 ±0.6 and for the latter was 5.4
± 1.5. The difference between the control smokers and nonsmokers was significant (p < 0.001). The difference between
the patients and control nonsmokers was not significant (p >
0.3).
1.5a±
0.6±
2.8"
Mean ±S.D.
DISCUSSION
It is currently unknown precisely what the SCE assay deter
mines (7, 20). Of the 4 chromosome instability or DNA repair
deficiency syndromes, Fanconi's anemia, Bloom's syndrome,
ataxia telengiectasia,
and xeroderma pigmentosum,
only
Bloom's syndrome is associated with increased spontaneous
SCE incidence (2, 5, 7, 9, 20). This increase in SCE frequency,
therefore, cannot be explained only on the basis of known DNA
repair mechanism (20). Some agents, such as irradiation, that
consistently cause chromatid and chromosomal breaks do not
cause increased SCE. On the other hand, in the in vitro system,
a concentration of chemicals that does not cause a significant
number of breaks consistently raised the SCE frequency (15).
In this regard, the SCE assay has been accepted widely as a
more sensitive system to screen mutagens compared to break-
JULY 1982
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2907
S. /noue ef a/.
age analysis. Furthermore, in a Chinese hamster cell culture,
the SCE incidence and the cell mutation rate proportionately
increased with the progressively increasing doses of chemicals
(1).
We believe that the SCE assay is, in spite of the disadvan
tages, the best and simplest assay system to test our hypoth
esis. In this study, we found no patient with a significantly
increased SCE frequency over that of controls. Although the
mean age of controls (all adults) is much higher than that of
patients, it was shown previously that sex and age have little
influence on the SCE levels (5). As was reported by other
investigators (8), control group smokers (more than a pack a
day) exhibited a higher SCE rate compared to the nonsmokers.
The past repetitious exposures to the chemotherapeutic drugs
and craniospinal irradiation have not led to any increased
frequency compared to the controls. This observation is con
sistent with the findings reported by other workers (8, 14, 17).
Otter ef al. (14) found an SCE frequency significantly higher
than in controls in children with ALL before the beginning of
treatment and while they were receiving the chemotherapy.
However, in 4 ALL long-term survivors who were off treatment,
their frequencies were comparable to the controls. Lambert ef
al. (8) reported normal frequencies in the patients treated with
5-fluorouracil,
vincristine,
Adriamycin,
cyclophosphamide,
bleomycin, thioTEPA, methotrexate, and prednimustine. Ra
posa (17) showed that cyclophosphamide treatment alone, or
in combination with other drugs, induced an increased inci
dence, but the frequency returned to the pretreatment level
after 10 days.
Our findings on SCE are in sharp contrast to the chromo
somal breakage study reported by Miller ef al. (10). These
authors reported a highly increased frequency in children who
had been treated with chemo- or radiotherapy for primary
cancers including leukemia, compared to their own pretherapy
frequencies and the frequencies of their parents and siblings.
The most prominent type of abnormality was translocation.
Although the chemotherapeutic regimens and radiation dose
used in these patients are not comparable to those in our
series, a portion of their study population, namely the children
with ALL, received roughly the same drug regimens as ours.
The ALL patients like other patients in the series of Miller ef al.
showed a much higher frequency of chromosomal aberration
compared to the controls. An interesting finding in their report
is that, while patients with Wilm's tumor and other cancers
showed predominently translocation, aberrations in ALL pa
tients were mainly simple deletions. The authors also showed
persistence of the high frequency of aberrations as late as 11
years after cessation of chemo- and/or radiotherapy, although
the diagnosis of these patients with persistent aberrations was
not mentioned. This apparent difference in results between our
study and the study done by Miller ef al. may be due to the
difference in the methods used (SCE versus breakage). The
study of Miller ef al. included solid-tumor patients in more than
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50% of the subjects, and this difference in the patient popula
tion studied also may account for the discrepancy in the results.
None of the patients in our study relapsed nor developed any
cancer thus far. The question of whether patients with an
increased SCE incidence, if found, may be at an increased risk
of developing cancer could be answered by only a large-scale
prospective study of a long duration.
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RESEARCH
VOL. 42
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Normal Sister Chromatid Exchange Frequency in Long-Term
Survivors with Acute Leukemia
Susumu Inoue, Laura Brown, Yaddanapudi Ravindranath, et al.
Cancer Res 1982;42:2906-2908.
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