Micronucleus Assay
as Radiosensitivity Indicator
in Head and Neck Tumor Patients.
Retrospective and Prospective Study
Bustos, E.; Di Giorgio, M.; Sardi, M.;
Aguilar Paredes, J. and Taja, M.R.
Presentado en: “Annual Meeting of the American Academy of Otolaryngology-Head and Neck Surgery”.
San Diego, USA, 21-25 setiembre 2002
MICRONUCLEUS ASSAY AS RADIOSENSITIVITY INDICATOR
IN HEAD AND NECK TUMOR PATIENTS. RETROSPECTIVE
AND PROSPECTIVE STUDY
1
2
1-3
1
Bustos, E. ; Di Giorgio, M. ; Sardi, M. ; Aguilar Paredes, J . and Taja, M.R.
2
1
2
Hospital Italiano
Nuclear Regulatory Authority
3
Mevaterapia
Argentina
INTRODUCTION
The aim of radiation oncologists is uncomplicated loco-regional control of cancer by radiation
therapy. The maximum dose used is limited by occurrence of severe late normal tissue
reactions (NTR) in the treatment area.
Patients receiving identical radiation treatments have different impacts on normal tissues,
varying from undetectable to sever.
The main goal is to anticipate the different NTR between patients in order to individualize their
treatment [1-4]. Nevertheless, the results obtained during the development of our research allow
us to establish that is necessary to accumulate 2 Gy equivalent whole body dose, to obtain a
predictive result.
Intrinsec radiosensitivity testing is considered more feasible and promising in normal than tumor
tissues because normal cell populations are less heterogeneous than tumor cell populations,
and clinical studies on radiosensitivity of normal tissue are easier to implement [5].
Cellular and molecular mechanisms of these reactions are not yet understood. There is
evidence that the extent of late NTR may depend on the individual cellular radiosensitivity.
The cytokinesis-blocked micronucleus assay is an established cytogenetic technique to
evaluate intrinsic cell radiosensitivity in tumor cells and lymphocytes and used for in vitro studies
in lymphocytes from patients undergoing radiotherapy in order to determine the clastogenic
effect induced by the treatment [6-7].
The objective has been to assess the individual cytogenetic response to radiotherapy applying
the cytokinesis blocked micronucleus (MN) assay to peripheral blood lymphocytes in two groups
of H&N cancer patients (retrospective and prospective group), in comparison with the observed
clinical response.
MATERIALS AND METHODS
Retrospective evaluation:
Nineteen patients with H&N tumors, undergoing radiation therapy as part of their oncological
protocol with and without late NTR, were retrospectively analyzed. Blood samples were
obtained from patients 6 to 18 month after the completion of the radiotherapy.
Spontaneous MN frequencies and radiation induced MN frequencies, after in vitro irradiation
with 2 Gy of Co-60, were evaluated for each patient.
Cytogenetic data, comparing expected MN frequencies, derived from the laboratory calibration
curve from healthy donors [8], with values observed after in vitro irradiation were analyzed using
2
χ test. Values > 3,84 (DF = 1; p < 0.05) indicate statistically significant differences between
observed and expected MN frequencies and thus, individual radiosensitivity or radioresistance.
The NTR inside the total irradiated volume was taken into account and correlated with the
individual cytogenetic response.
305
Prospective evaluation:
Fifteen patients with H&N and cervix tumors, undergoing radiation therapy as part of their
oncological protocol, were prospectively analyzed; the blood for MN assay was obtained from
the patients during fractionated therapy. Blood samples were taken just before treatment, midway during treatment and /or on completion of treatment.
The cytogenetic data were analyzed using a mathematical model to evaluate the attenuation of
the cytogenetic effect as a function of the time between a single exposure and blood sampling,
estimating a cytogenetic recovery factor (k) and its correlation with the individual radiosensitivity.
F(MN)=∑( MNi . e
–di.k
)
Catena formula [9]
F(MN): cytogenetic effect measured in lymphocytes of patients during therapy
MNi: cytogenetic effect calculated for each day of treatment
di: number of days between an exposure and blood sampling
k: cytogenetic recovery factor
If k tends to 0, F(MN) approaches the in vitro calibration curve, implies
decreasing recovery from the cytogenetic effect
Increasing values of k are directly proportional to increasing recovery from the
cytogenetic effect as a function of time
RESULTS
In the retrospective evaluation, lymphocytes from 3 of the 4 patients that had developed late
reactions were significantly more radiosensitive than lymphocytes from the rest of the patients
and normal donors (individuals without cancer). The individual cytogenetic response (in vitro
normal tissue radiosensitivity testing) suggests a correlation with the maximum grade of late
reaction (osteonecrosis, fibrosis and trismus). Figure 1. and table 1.
Table 1. Spontaneous MN frequencies and radiation induced MN frequencies,
2
after in vitro irradiation with 2 Gy of Co-60, analyzed using χ test
Patient
Dose = 0Gy
Observed
Frecuency
1
Dose = 0Gy
2
2
χ
Test
Observed
Frecuency
χ
Test
0.0423
36.15
0.3412
134.06
2
0.0095
3.52
0.1694
1.17
3
0.0216
1.05
0.1984
1.11
4
0.0128
1.15
0.2012
1.59
5
0.0132
0.97
0.2053
2.44
6
0.0138
0.70
0.1240
19.60
7
0.0398
29.26
0.3450
140.62
8
0.0119
1.71
0.2041
2.18
9
0.0102
2.91
0.1358
12.67
10
0.0136
0.79
0.1600
3.15
11
0.0123
1.45
0.1780
0.20
12
0.0098
3.27
0.1920
0.34
13
0.0200
0.42
0.2082
3.15
14
0.0080
5.00
0.1478
7.15
15
0.0136
0.80
0.1319
14.82
16
0.0112
2.15
0.1346
13.30
17
0.0134
0.88
0.1369
12.10
18
0.0272
5.71
0.1790
0.14
19
0.0287
7.51
0.3272
111.23
306
Fig. 1. Comparison of cytogenetic data with the dose effect curve, after 2 Gy in vitro irradiation
of blood samples from H&N cancer patients (retrospective analysis)
MN Frequency
0,4
0,3
0,2
0,1
0
0
1
Dose [Gy]
2
3
In the prospective evaluation, a significant difference between patients´ data and the
calibration curve was found above 2 Gy of equivalent whole body dose.
Factor (k) correlated with the individual radiosensitivity. Patients with low recovery from the
cytogenetic effect (k tending to zero) developed late toxicity (fibrosis, trismus and actinic
rectitis). Figure 2. and table 2.
Table 2. Cytogenetic data and cytogenetic recovery factor k for 15 patients undergoing
radiotherapy to pelvic and head & neck sites
Patient Nº of fractions
Equivalent dose
F (MN)
[Gy]
observed
F (MN)
Calibration curve
K
Cytogenetic recovery factor
1
10
1.555
0.117
0.127
0.036
2
10
1.507
0.087
0.122
0.097
3
10
1.256
0.081
0.096
0.096
4
10
1.450
0.137
0.116
0.001
5
10
1.127
0.083
0.083
0.083
6
10
1.392
0.078
0.109
0.112
7
10
1.589
0.100
0.131
0.075
8
10
1.510
0.107
0.122
0.054
9
15
2.149
0.094
0.205
0.106
10
23
4.728
0.189
0.774
0.029
11
15
2.730
0.124
0.301
0.083
12
15
2.469
0.112
0.256
0.088
13
24
2.911
0.339
0.359
0.015
14
31
2.209
0.128
0.215
0.048
15
35
3.714
0.149
0.506
0.037
307
Fig. 2. Comparison between cytogenetic data of patients with our calibration curve (MN/CB)
1
Cervix Cancer Patients (1)
Cervix Cancer Patients (2)
Head & Neck Cancer Patients
MN/CB
0,8
0,6
0,4
0,2
0
0
1
2
3
Equivalent Dose [Gy]
4
5
CONCLUSIONS
The cytogenetic data demonstrate that the cytokinesis blocked micronucleus assay is suitable
for the retrospective evaluation of individual cytogenetic response to radiotherapy. The assay
applies well to the objective. It is easy and rapid to perform in a routine clinical laboratory.
It seems that the micronucleus assay can be used as a radiosensitivity indicator when the
irradiated volume involves parts of the body having a large blood volume and flow, representing
at least 10 % of the total corporal volume or compromising bone marrow.
In the retrospective evaluations, both, spontaneous and radiation induced (2Gy) micronucleus
frequencies were significantly increased, compared with the expected values from the
calibration curve, in those patients who had developed late tissue reactions. This
hypersensitivity to radiation may be due to a lack of repair of DNA damage.
The retrospective evaluation with micronucleus assay would allow predicting the late toxicity, in
patients that would require re-irradiation treatment.
The correlation obtained encourages us to begin a prospective study, using MN assay, in order
to predict late effects of radiation therapy in patients with head and neck malignant tumors. The
potential benefits of detecting patients with a high probability of suffering severe late reactions
are significant in clinical radiotherapy.
In the prospective evaluations, in vitro irradiation of the patient blood samples before radiation
therapy was not predictive of the individual cytogenetic response.
In the same group of patients prospectively studied, as well as in a cervix cancer patient group,
previously investigated, the cytokinesis blocked micronucleus assay (in vitro) had a good
correlation with the clinical late toxicity (in vivo) above 2 Gy equivalent whole body dose, in
agreement with other studies [10]. There was not correlation with clinical acute toxicity.
After therapeutic irradiation a low value of k indicates high radiosensitivity of the lymphocyte pool
and thus, a patient with low recovery from the cytogenetic effect (patient 4 and 13. Table 2.)
308
These studies would contribute with radiosensitivity tests of current use but applying a rapid
methodology easy to implement in a routine clinical laboratory. Long-term clinical observations
could confirm the validity of k in expressing predisposition of the subject to develop delayed
effects.
The identification of radiosensitive patients, through MN test, will allow the individualization of
patient treatment. Normal tissue radiosensitivity testing could be used to dose-escalate or dosereduce without compromising tumor control rates.
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