- Email: [email protected]
Time-course of radiation-induced chromosomal aberrations in tumor patients after radiotherapy
Int. J. Radiation Oncology Biol. Phys., Vol. 63, No. 4, pp. 1214 –1220, 2005 Copyright © 2005 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/05/$–see front matter
doi:10.1016/j.ijrobp.2005.03.056
CLINICAL INVESTIGATION
Normal Tissues
TIME-COURSE OF RADIATION-INDUCED CHROMOSOMAL ABERRATIONS IN TUMOR PATIENTS AFTER RADIOTHERAPY IRENE MÜLLER,* HANS GEINITZ, M.D.,† HERBERT BRASELMANN,* ADOLF BAUMGARTNER, PH.D.,* ANNETTE FASAN,* REINHARD THAMM,† MICHAEL MOLLS, M.D.,† VIKTOR MEINEKE, M.D.,‡ AND HORST ZITZELSBERGER, M.D.* *GSF-National Research Center for Environment and Health, GmBH, Institute of Molecular Radiation Biology, Neuherberg, Germany; †Department of Radiation Oncology, Klinikum Rechts der Isar, Technical Univeristy, Munich, Germany; ‡Institute of Radiobiology, German Armed Forces, Munich, Germany Purpose: Radiation-induced chromosome aberrations are routinely used in biologic dosimetry to monitor radiation exposure. Translocations are considered stable aberrations with time after exposure. This study was performed to determine the temporal persistence of radiation-induced translocations during a 36-month period in therapeutically irradiated testicular seminoma patients who underwent partial body exposure (>10% of bone marrow). Methods and Materials: Chromosome analyses were carried out in peripheral lymphocytes of 11 patients with testicular seminoma (n ⴝ 9), germinoma (n ⴝ 1), or follicular non-Hodgkin’s lymphoma (n ⴝ 1). All patients received radiotherapy with photons from a linear accelerator; in 1 case, additional electron beams were used. Doses ranged from 26 Gy (seminoma) to 45 Gy (non-Hodgkin’s lymphoma). None of the patients received chemotherapy. From each patient, blood samples were taken during the 36 months after irradiation at defined points. Chromosomal aberrations were scored after fluorescence in situ hybridization painting of chromosomes 1, 4, and 12 in combination with a pancentromeric probe. Results: For 9 patients (7 with testicular seminoma, 1 with germinoma, and 1 with non-Hodgkin’s lymphoma), a significant temporal decline of translocations, with a mean decline rate of 4.4% ⴞ 0.4% monthly, could be detected. Two testicular seminoma patients showed no temporal decline of aberration frequencies. Conclusion: Most partial body irradiated patients (9 of 11) showed a significant temporal decline of translocation frequencies during a 36-month period. Thus, reciprocal translocations after partial body irradiation cannot be regarded as stable over time. The temporal decline of aberration frequencies has to be taken into account for retrospective dose estimations. © 2005 Elsevier Inc. Radiation induced chromosome aberrations, Translocations, Fluorescence in situ hybridization, Temporal decline, Biodosimetry.
INTRODUCTION Radiation-induced chromosomal aberrations can be detected and quantified in peripheral lymphocytes many years after exposure to ionizing irradiation using fluorescence in situ hybridization (FISH) with whole chromosome painting probes (for review, see Bauchinger [1] and Natarajan and Boei [2]). This so-called FISH-painting technique is usually used for retrospective dose estimations and mechanistic investigations on chromosome aberration formation after radiation exposure. For these purposes, a partial genome analysis using combinations of whole chromosome probes for three chromosomes in one or more colors is usually
performed. Recently, techniques for whole genome analyses of radiation-induced chromosome aberrations have also become available (3– 6). However, they are far too time consuming for routine analysis. In biodosimetric applications, translocations occurring in peripheral lymphocytes are considered stable markers of radiation exposure, assuming that they do not decline with time after exposure. This is an important prerequisite for dose reconstructions of past exposures. However, no extensive data have demonstrated the persistence of radiation-induced translocations with time after exposure. In the studies performed so far that have investigated animals (7, 8), victims of accidental irradiation (9 –12), or in vitro irradiated cells (13, 14), contradicting
Reprint requests to: Horst Zitzelsberger, Ph.D., GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Institute of Molecular Radiobiology, Ingolstädter Landstr. 1, Neuherberg D-85764 Germany. Tel: (⫹49) 89-318-73-421; Fax: (⫹49) 89318-72-873; E-mail: [email protected] Supported financially by the Federal Office of Defense Tech-
nology and Procurement, Grant E/B41G/Z0531/Z5803. H. Braselmann and H. Geinitz contributed equally to this study. Acknowledgments—The skillful technical assistance of S. Schröferl and E. Konhäuser is gratefully acknowledged. Received Jan 27, 2005, and in revised form Mar 24, 2005. Accepted for publication Mar 24, 2005. 1214
Temporal decline of radiation-induced translocations
results have been obtained. In another study, the examined cohort of therapeutically irradiated patients was too heterogeneous to receive clear results on the temporal stability of translocations (15). Some of these studies have shown that the translocation frequencies are stable over time (9, 11, 15), and others detected a decline in translocation frequencies (7, 12, 14). The aim of the present study was to evaluate the time course of translocation frequencies in peripheral blood lymphocytes after therapeutic partial body irradiation. We present a data set of patients who received pure radiotherapy (RT) either as the sole treatment for their malignancy or as adjuvant treatment. Translocations were prospectively evaluated before treatment and at defined points up to 36 months after treatment. Decline curves could be calculated for each individual patient.
● I. MÜLLER et al.
1215
approved this study. All patients provided written informed consent before participating.
Cell culture Blood samples were obtained between 1999 and 2003. For each sample, whole blood cultures were set up with heparinized peripheral blood. We added 0.5 mL of blood to each 4.5 mL of Roswell Park Memorial Institute medium 1640 plus Glutamax (Gibco) supplemented with 15% fetal calf serum, 2.5% phytohemagglutinin, antibiotics, and bromodeoxyuridine (at a final concentration of 9.6 ⫻ 10⫺6 g/mL). Cultures were incubated for 45 h at 37°C under 5% carbon dioxide-containing atmosphere followed by a Colcemid treatment (0.1 g/mL) for 3 h under the same conditions. Subsequently, chromosome preparation was performed according to standard procedures (17). The slides were stored at ⫺20°C under nitrogen atmosphere until use.
Fluorescence in situ hybridization METHODS AND MATERIALS Patients Chromosome analyses were performed in 11 male cancer patients who exclusively received RT either as the sole treatment or as adjuvant treatment. No concomitant or sequential chemotherapy was applied. From each patient, at least 10% of the blood cellproducing bone marrow had been irradiated during therapy. Moreover, the survival of each patient was assumed to be ⬎2 years. None of the patients had received any RT or chemotherapy before their current treatment or during follow-up. Nine patients (age 32– 65 years at the beginning of the study) received adjuvant RT after one-sided inguinal orchiectomy for Stage I testicular seminoma. Treatment was given by ventrodorsal opposed fields to a total dose of 26 Gy in 2-Gy fractions, 5 times weekly. The fields extended from the upper border of the 11th thoracic vertebra to the lower border of the 4th lumbar vertebra. Laterally, the fields extended to the edges of the transverse spinal processes. Approximately 12% of the active bone marrow was included in the radiation fields (16). One patient (aged 22 years) received RT for germinoma in the pinealis region. The whole craniospinal axis was treated to a dose of 24 Gy in 1.6-Gy fractions, 5 times weekly. The neurocranium and upper cervical spine were irradiated using two lateral opposed fields, and the remaining spine, including the third sacral vertebra, was treated by two adjoining dorsal fixed fields. After treatment of the craniospinal axis, a boost to the tumor region was applied with a dose of 16 Gy in 1.6-Gy fractions. Approximately 42% of the active bone marrow was included in the radiation fields in this patient (16). One patient (aged 46 years) received definitive RT for follicular non-Hodgkin’s lymphoma Stage Ia of the right inguinal lymph nodes. The whole abdomen and pelvis (“abdominal bath”) were treated with a dose of 25.5. Gy in 1.5-Gy fractions, 5 times weekly. Treatment was done with two ventrodorsal opposed fields. After 12 Gy, the kidneys and part of the liver were shielded. The right groin was treated with electrons to a dose of 45 Gy and the left groin to a dose of 25.2 Gy, both in 1.8-Gy fractions, 5 times weekly. Approximately 50% of the active bone marrow was included in the radiation fields in this patient (16). For each patient, blood samples were taken before RT, immediately after RT, and 2, 5, 8, 12, 18, 24, 30, and 36 months after RT. The ethics committee of the Technical University Munich
Whole chromosome painting probes for chromosomes 1, 4, and 12 were used in combination with a pancentromeric DNA probe. Plasmid DNA of chromosome-specific Hind III pBS libraries of chromosomes 1, 4, and 12 (18) was isolated, as described previously (19). Plasmid DNA samples were labeled with either biotin (chromosomes 1 and 4) or digoxigenin (chromosomes 4 and 12) using nick-translation, as described by Weier et al. (20). A pancentromeric probe was generated and biotinylated by polymerase chain reaction according to Bauchinger et al. (21). After hybridization for 24 – 48 h, bound biotin-labeled probes for chromosomes 1 and 4 and the biotinylated pancentromeric probe were detected with fluorescein isothiocyanate (FITC)streptavidin conjugate (Dianova, Hamburg, Germany) and bound digoxigenin-labeled whole chromosome painting probes for chromosomes 4 and 12 with Cy3-labeled antibodies (Dianova, Hamburg, Germany). Counterstaining was performed with 4=,6diamidinophenylindol (DAPI) in antifade solution (Vecta Shield, Linaris GmbH, Wertheim-Bettingen, Germany) at a concentration of 1 g/mL. To perform chromosome analyses only in the first division metaphases, FISH painting was combined with fluorescence plus Giemsa staining, as described by Kulka et al. (22).
Chromosome analysis After FISH painting, chromosome 1 appeared in green (FITC), chromosome 4 in orange (combination of FITC and Cy3), and chromosome 12 in red (Cy3). The centromeres were additionally stained in green (FITC). Because the FISH signals for the centromeric regions are very bright, they were also visible on chromosomes 1 and 4. Counterstaining of all chromosomes appeared in blue (DAPI). The PAINT (Protocol for Aberration Identification and Nomenclature Terminology) nomenclature system (23), as well as the S&S nomenclature system (24 –26) was applied to describe the observed painting patterns. All images of metaphases exhibiting aberrations of the painted chromosomes were acquired and stored using the ISIS image analysis system (Metasystems, Altlussheim, Germany). For an analysis of the time course of aberrations, reciprocal translocations t(Ab) ⫹ t(Ba) and one-way patterns t(Ba) ⫹ ace(b), t(Ab), and t(Ba) were considered. This corresponds to 2B and 2B I-III according to the S&S nomenclature system. In addition, dicentric chromosomes dic(BA) ⫹ ace(ba), dic(BA) ⫹ ace(b),
Volume 63, Number 4, 2005
Fig. 1. Translocation rates in 11 therapeutically irradiated patients immediately and 2 months after radiotherapy. Error bars indicate standard error of mean for each patient. Two cases (Patients 4 and 9) had significant differences in translocation rates between two time points (asterisks).
1433 (21/9) 555 (14/14)* 1669 (23/18) 1062 (20/15) 1115 (20/3) 833 (41/31) 773 (21/9) NA 1038 (20/11) 1259 (21/14) 355 (12/4)* 1033 (24/14) 442 (11/9)* 1027 (20/27) 661 (22/20) 2011 (22/17) 428 (21/17) NA 585 (20/22) 869 (20/15) 749 (9/19)* 999 (38/19) 1767 (19/18)* 439 (12/7)* 984 (21/10) 661 (24/17) 1181 (21/15) 1151 (20/15) 573 (20/17) 445 (24/22) 514 (8/17)* 886 (20/9) 560 (26/11)
30 mo after RT 24 mo after RT
ace(ba), and dic(BA), corresponding to 2A and 2A IV-VI according to the S&S nomenclature, were taken into account and could be distinguished using the pancentromeric FISH signals.
Statistical analysis Abbreviations: Pt. no. ⫽ patient number; RT ⫽ radiotherapy; NA ⫽ no samples available. * Scoring criteria could not be completely fullfilled owing to limited numbers of slides.
666 (22/18) 480 (22/16) 694 (20/7) 350 (27/20) 1253 (23/15) 442 (15/22)* 411 (21/23) 297 (22/21) 1441 (20/18) 587 (20/11) 504 (39/24) 1918 (0/0) 2027 (2/0) 2623 (0/1) 2107 (1/0) 2139 (0/0) 2253 (0/0) 2051 (0/0) 1960 (0/0) 2106 (1/1) 2064 (1/0) 2300 (1/0) 2 4 5 6 7 8 9 10 11 13 14
376 (20/12) 429 (20/17) 601 (30/24) 191 (24/18) 1023 (33/19) 691 (20/28) 645 (23/19) 770 (37/44) 878 (24/14) 627 (30/22) 285 (69/43)
481 (23/19) 218 (20/25) 574 (37/25) 196 (39/25) 1562 (36/21) 640 (20/25) 257 (21/22) 522 (33/31) 854 (21/10) 378 (23/17) 210 (34/41)
808 (20/17) 298 (20/16) 341 (20/15) 242 (26/20) 781 (38/12) 598 (32/30) 601 (20/29) 466 (32/11) 1029 (20/20) 398 (20/10) 298 (23/31)
735 (20/11) 454 (21/16) 900 (32/32) 612 (38/40) 1889 (32/35) 1430 (42/31) 1002 (26/21) 654 (30/11) 1649 (23/37) 1557 (20/28) 898 (28/27)
18 mo after RT 12 mo after RT 8 mo after RT 5 mo after RT 8 wk after RT After RT Before RT Pt. no.
Cells scored (translocations/dicentrics) (n)
Table 1. Number of translocations and cells scored in 11 irradiated patients at different points
● Biology ● Physics
1421 (20/10) 652 (20/11) 1569 (20/8) 568 (13/6)* 1385 (11/7)* 655 (17/9)* 1303 (26/20) 1710 (24/11) 703 (20/13) 2042 (14/7) 2097 (30/19)
I. J. Radiation Oncology
36 mo after RT
1216
Calculations were performed using an exponential decline model, which was fitted by logarithmic-linear Poisson regression (27). For each patient, a mean infinitesimal decline rate, k (also known as time constant or hazard rate, percentage per month), and mean half-life, h [⫽ ln (2)/k] for translocations and dicentrics, were calculated using follow-up times of between 2 and 36 months after irradiation. Statistical analysis was performed using analysis of deviance (28) with a likelihood-ratio test for intercomparison of decline rates and chi-square goodness-of-fit test for investigation of deviations from the regression model.
RESULTS A total of 109,000 metaphases were scored from 11 therapeutically irradiated patients at 10 different times before and after RT. The criteria for translocation scoring have been established; either 2000 cells or 20 translocations (reciprocal or one-way) had to be scored for each blood sample to obtain sufficient results for additional statistical interpretation (Table 1). The time course of the aberrations was calculated for simple reciprocal and one-way translocations (2B and 2B I-III) and dicentrics with and without acentric fragment, as well as solitary acentric fragments (2A and 2A IV-VI). The initial translocation frequencies were plotted immediately after RT and, for comparison, 8 weeks after RT (Fig. 1). Translocation frequencies immediately after RT were greatest in the 2 patients treated with the highest radiation fields (Patient 6, craniospinal axis and Patient 14, abdominal bath; Fig. 1). Taking all follow-up stages between 8 weeks and 36 months after irradiation into account,
Temporal decline of radiation-induced translocations
Fig. 2. Calculated decline rates for translocations and dicentrics in 9 irradiated patients (7 with testicular seminoma, 1 with germinoma [Patient 6], and 1 with non-Hodgkin’s lymphoma [Patient 14]). *Decline rates for translocations and dicentrics of victim of Estonia radiation accident (whole body exposure, 12). Error bars indicate standard error for each decline rate.
we obtained average decline rates for translocations and dicentrics (Fig. 2). The parameters and statistics from the calculation (i.e., decline rates, extrapolated initial values, chi-square goodness-of-fit test, and half-life periods) are shown in Table 2. For Patients 8 and 11, both treated for testicular seminoma, no temporal decline for translocations could be observed for the complete period (Fig. 3b). In these patients, the initial translocation rates did not differ from the final rates, although a decrease in translocations rates was noted after 12 and 18 months, followed by an increase to the initial levels later. For the other 9 patients, a significant temporal decline
● I. MÜLLER et al.
1217
became apparent for translocations, as well as for dicentrics (Fig. 2). The mean infinitesimal decline rate for translocations in these patients was 4.4%/mo (range, 2.4 – 6.9%). This complies with a half-life of 15.6 months (95% confidence interval, 13.3–18.9). No significant differences in the decline rates of these 9 patients could be detected (likelihood-ratio test, p ⬎ 0.05). An example (Patient 6) of a temporal decline in translocation rates is demonstrated in Fig. 3a. Patients 7, 8, 9, 13, and 14 had significant deviations from a uniform time course (chi-square goodnessof-fit test, p ⬍ 0.05). For Patient 7, the rate of translocations 5 months after irradiation was greater than average. Patients 9 and 13 showed irregular deviations at different times, and Patient 14 exhibited a fast decline within the first year and after 30 months. In Patient 8, reduced rates of translocations were detected during the second year, but the final rates of translocations corresponded to the initial rates.
DISCUSSION The aim of this study was to investigate the time course of translocation frequencies after partial body exposures using FISH painting. Because FISH painting for translocation scoring has been regarded as the gold standard endpoint for radiation biologic dosimetry (29), it appears to be essential to determine the persistence of radiation-induced translocations over time. To date, some studies of whole body exposures after radiation accidents have reported that radiation-induced translocations persist with time (9, 11, 15), although other studies have demonstrated results with a significant decline of translocation frequencies (7, 12, 14). However, in several of these studies, either the initial translocation rates after irradiation were not available or the radiation conditions of the individuals were very inhomo-
Table 2. Statistical analysis (logarithmic-linear Poisson regression) of translocation rates from 8 weeks to 36 months in 11 irradiated patients
Pt. no. 2 4 5 6 7 8 9 10 11 13 14 Mean, without patients 8 and 11
Age (y) 43 65 44 22 32 34 34 39 36 35 46
Malignancy Seminoma Seminoma Seminoma Germinoma Seminoma Seminoma Seminoma Seminoma Seminoma Seminoma NHL
Extrapolated initial values (per 100 cells)
Mean infinitesimal* decline rate k (% mo)
Mean half-life (mo)
Chi-square goodnessof-fit test
3.11 6.75 5.86 15.32 2.75 3.11 4.70 8.11 1.59 4.57 10.44
3.07 ⫾ 1.1 3.38 ⫾ 0.9† 4.92 ⫾ 0.6‡ 6.92 ⫾ 1.1‡ 3.09 ⫾ 1.4‡ ⫺0.06 ⫾ 1.4 2.43 ⫾ 1.1§ 4.67 ⫾ 0.7‡ ⫺0.82 ⫾ 0.8 5.14 ⫾ 1.5‡ 5.43 ⫾ 1.3‡ 4.43 ⫾ 0.39‡
22.6 20.5 14.1 10.0 22.4 — 28.5 14.8 — 13.5 12.8 15.6
12.41 7.26 5.44 11.07 23.04‡ 25.66‡ 12.48§ 4.98 7.14‡ 21.60‡ 26.12‡ 156.5‡
†
Abbreviations: Pt. no. ⫽ patient number; df ⫽ degrees of freedom; NHL ⫽ non-Hodgkin’s lymphoma. * In short intervals ⌬t (⬍1 mo), translocation frequency decreases by k⌬t%. † p ⬍ 0.01. ‡ p ⬍ 0.005. § p ⬍ 0.05.
df 6 6 6 6 6 6 5 5 6 6 6
1218
I. J. Radiation Oncology
● Biology ● Physics
Fig. 3. Time course of radiation-induced translocations in 3 patients. (a) Patient 6 showing decline of translocation frequencies during 36-month period. (b) Patients 8 and 11 showing final unchanged translocation rates after 36 months. Error bars indicate standard error of mean for each translocation rate.
geneous (15), making clear interpretation of the data difficult and possibly explaining the reported discrepancies. Our investigations of therapeutically irradiated patients represent the first study of a largely homogeneously exposed cohort (9 seminoma patients) that included regular blood sampling for each patient to generate decline curves and examine the time course of translocation frequencies after partial body exposure. Published data from therapeutically irradiated patients are also rare. Only a very few studies of the time course of translocation frequencies from partial body irradiated patients have been published (30 – 32). Gebhart et al. (30) reported on a decline of radiationinduced translocations in an inhomogeneous group of patients after several doses and treatment volumes. In another, more homogeneous, cohort of breast cancer patients, the results were not meaningful owing to the small portion of bone marrow in the radiation field (⬍5%), resulting in a nonstatistically significant tendency of a temporal decline in
Volume 63, Number 4, 2005
translocation rates (31). The third study demonstrated a temporal decline in testicular seminoma patients; however, no individual time courses of translocation frequencies at defined times after RT for each patient, and thus no decline curves or statistically evaluated decline rates of translocation frequencies, could be established (32). In our study, individual time courses of translocation frequencies from a largely homogeneously irradiated cohort of patients (9 with testicular seminoma and comparable irradiation conditions, 1 with germinoma, and 1 with nonHodgkin’s lymphoma) could be demonstrated, including the initial translocation rates. As demonstrated in Fig. 2 and exemplary in Fig. 3, only in 2 exposed patients did the translocations persist for a 36-month period, and in 9 exposed patients, a clear decline became apparent, with a mean half-life of 15.6 months. Moreover, the observed dicentric and translocation rates were not significantly different for all patients in our cohort. A reference case from the Estonia accident (12) showed a significantly lower decline rate for translocations than for dicentrics (Fig. 2). This disagreement was probably a result of the different irradiation conditions, which consisted of whole body exposure at a lower dose for the Estonia case and of partial body exposure at high doses for our patient cohort. For a comparison of the half-lives observed in our patient cohort, we reviewed a previous study on dicentrics that had been scored after fluorescence plus Giemsa staining in peripheral lymphocytes from therapeutically irradiated testicular seminoma patients (33). In that study, an initial half-life of about 0.4 years was calculated for dicentrics (33). The unexpected steep decline of translocation rates, with a mean half-life of 15.6 months (Table 2), in the present patient cohort might have resulted from the partial body exposure with high doses of ionizing radiation. Because the half-life of peripheral lymphocytes is limited and a mean life span of 1574 days was estimated (34), cells exhibiting both aberration types, translocations and dicentrics, decline. In contrast, the repopulation of peripheral lymphocytes carrying translocations should be not affected, although dicentrics will disturb cell division of precursor cells and thus the repopulation of lymphocytes. The observation of a decline of peripheral lymphocytes carrying translocations in our patient cohort could be partly attributed to additional lethal aberrations (other than the scored reciprocal translocations) that were present in the same cell. It might, therefore, be argued that translocation scoring in so-called stable cells (i.e., cells that contain only transmissible aberrations during cell cycle) would have resulted in long-term persistence of translocation rates, as demonstrated by Lindholm and Edwards (11). Such “stable” cells have been characterized by also scoring the unpainted part of the genome for the presence of unstable aberrations. This approach is difficult to perform because undetectable complex aberrations that are not transmissible during the cell cycle might also be present in the unpainted part in addition to the dicentrics, rings, and acentrics considered by Lindholm and Edwards (11). We have tried such an analysis in selected blood samples and
Temporal decline of radiation-induced translocations
failed because almost every cell with a translocation carried additional unstable aberrations in the unpainted chromosomes. Additionally, it seems not to be justified for statistical reasons to perform translocation analyses only in “stable” cells, because the same scoring criteria must then be applied to metaphases without translocations in the painted part of the genome. Such a scoring procedure appears not to be feasible if high cell numbers of many individuals must be scored. Moreover, it is evident that not only simple dicentrics in the unpainted part of the genome renders a cell “unstable,” but also complex aberrations may cause a nontransmissible cell during a cell cycle that is not scored in the counterstained part in a FISH-painted metaphase. For a demonstration of the time course of translocation and dicentric frequencies, an exponential model was used for each patient. This resulted in individual decline curves and calculated decline rates (Table 2 and Figs. 2 and 3). Patients 6 and 14 showed the greatest aberration frequencies, in accordance with the larger radiation fields and higher doses applied in these patients. Despite these greater aberration frequencies, a significant deviation from the mean decline rate and the mean half-life of translocations of testicular seminoma patients could not be observed. For 2 patients (Patients 8 and 11), final unchanged translocation rates could be observed after 36 months, although reduced rates became apparent in the second year after irradiation followed by increasing translocation frequencies up to the initial level. This phenomenon could be explained by individually different time courses of lymphocyte repopulation or by persisting irradiated lymphocytes that reenter the blood circulation at a certain point after irradiation. A different volume of active bone marrow in the radiation field
● I. MÜLLER et al.
1219
could be excluded as an explanation for these 2 cases exhibiting final unchanged translocation rates, because the number of vertebrae in the radiation fields was identical among the testicular seminoma patients. A possible explanation for this observation is again the presence of individual cell kinetics of repopulating blood cells from stem cells or the circulation of damaged cells between different lymphocyte-containing compartments. According to a model described by Trepel (35) and Wuestermann and Cronkite (36), lymphocytes are believed to recirculate among the central blood compartment, spleen, lymph nodes, and peripheral tissues, such as the thymus and lymphoid tissue of the gut. Another possibility to explain this phenomenon in our study is the finding that different T-lymphocyte clones exhibit a different radiosensitivity (37) and a different recovery after irradiation (38). Assuming that interindividual variations are present, such observations also may influence the time course of aberration-carrying lymphocytes after therapeutic irradiation. CONCLUSION We investigated 11 therapeutically irradiated patients during a 36-month period for radiation-induced translocation frequencies. We observed a significant temporal decline in translocation frequencies in 9 patients. In 2 patients, the translocation frequencies persisted within this period. The decline of radiation-induced translocations with time might have been a result of the partial body exposure in the high-dose range. The calculated decline rates have an impact on retrospective dose estimations, especially in the high-dose range of partial body irradiation.
REFERENCES 1. Bauchinger M. Retrospective dose reconstruction of human radiation exposure by FISH/chromosome painting. Mutat Res 1998;404:89 –96. 2. Natarajan AT, Boei JJ. Formation of chromosome aberrations: Insights from FISH. Mutat Res 2003;544:299 –304. 3. Schrock E, du Manoir S, Veldman T, et al. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494 – 497. 4. Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12:368 –375. 5. Greulich KM, Kreja L, Heinze B, et al. Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH. Mutat Res 2000;452:73– 81. 6. Loucas BD, Cornforth MN. Complex chromosome exchanges induced by gamma rays in human lymphocytes: An mFISH study. Radiat Res 2001;155:660 – 671. 7. Tucker JD, Breneman JW, Briner JF, et al. Persistence of radiation-induced translocations in rat peripheral blood determined by chromosome painting. Environ Mol Mutagen 1997; 30:264 –272. 8. Tucker JD, Marples B, Ramsey MJ, et al. Persistence of chromosome aberrations in mice acutely exposed to 56Fe⫹26 ions. Radiat Res 2004;161:648 – 655. 9. Salassidis K, Georgiadou-Schumacher V, Braselmann H, et al.
10. 11. 12. 13.
14.
15.
Chromosome painting in highly irradiated Chernobyl victims: A follow-up study to evaluate the stability of symmetrical translocations and the influence of clonal aberrations for retrospective dose estimation. Int J Radiat Biol 1995;68:257–262. Lindholm C, Tekkel M, Veidebaum T, et al. Persistence of translocations after accidental exposure to ionizing radiation. Int J Radiat Biol 1998;74:565–571. Lindholm C, Edwards A. Long-term persistence of translocations in stable lymphocytes from victims of a radiological accident. Int J Radiat Biol 2004;80:559 –566. Bauchinger M, Schmid E, Braselmann H. Time-course of translocation and dicentric frequencies in a radiation accident case. Int J Radiat Biol 2001;77:553–557. Boei JJ, Vermeulen S, Natarajan AT. Detection of chromosomal aberrations by fluorescence in situ hybridization in the first three postirradiation divisions of human lymphocytes. Mutat Res 1996;349:127–135. Matsumoto K, Ramsey MJ, Nelson DO, et al. Persistence of radiation-induced translocations in human peripheral blood determined by chromosome painting. Radiat Res 1998; 149:602– 613. Tawn EJ, Whitehouse CA. Persistence of translocation frequencies in blood lymphocytes following radiotherapy: Implications for retrospective radiation biodosimetry. J Radiol Prot 2003;23:423– 430.
1220
I. J. Radiation Oncology
● Biology ● Physics
16. Ellis RE. The distribution of active bone marrow in the adult. Phys Med Biol 1961;5:255–258. 17. Huber R, Kulka U, Lorch T, et al. Technical report: Application of the Metafer2 fluorescence scanning system for the analysis of radiation-induced chromosome aberrations measured by FISH-chromosome painting. Mutat Res 2001;492: 51–57. 18. Pinkel D, Landegent J, Collins C, et al. Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 1988;85:9138 –9142. 19. Schmid E, Zitzelsberger H, Braselmann H, et al. Radiationinduced chromosome aberrations analysed by fluorescence in situ hybridization with a triple combination of composite whole chromosome-specific DNA probes. Int J Radiat Biol 1992;62:673– 678. 20. Weier HU, Lucas JN, Poggensee M, et al. Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations. Chromosoma 1991;100:371–376. 21. Bauchinger M, Schmid E, Zitzelsberger H, et al. Radiationinduced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe. Int J Radiat Biol 1993;64:179 –184. 22. Kulka U, Huber R, Muller P, et al. Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes. Int J Radiat Biol 1995;68: 25–27. 23. Tucker JD, Morgan WF, Awa AA, et al. PAINT: A proposed nomenclature for structural aberrations detected by whole chromosome painting. Mutat Res 1995;347:21–24. 24. Savage JR, Simpson P. On the scoring of FISH-“painted” chromosome-type exchange aberrations. Mutat Res 1994;307: 345–353. 25. Savage JR, Simpson PJ. FISH “painting” patterns resulting from complex exchanges. Mutat Res 1994;312:51– 60. 26. Simpson PJ, Savage JR. Dose-response curves for simple and complex chromosome aberrations induced by X-rays and de-
Volume 63, Number 4, 2005
27. 28. 29. 30. 31.
32.
33.
34. 35. 36.
37.
38.
tected using fluorescence in situ hybridization. Int J Radiat Biol 1996;69:429 – 436. Frome EL. The analysis of rates using Poisson regression models. Biometrics 1983;39:665– 674. Nelder JA, Wedderburn RWM. Generalized linear models. J R Statist Soc A 1972;135:370 –384. Tucker JD. Sensitivity, specificity, and persistence of chromosome translocations for radiation biodosimetry. Mil Med 2002; 167:8 –9. Gebhart E, Neubauer S, Schmitt G, et al. Use of a three-color chromosome in situ suppression technique for the detection of past radiation exposure. Radiat Res 1996;145:47–52. Huber R, Braselmann H, Kulka U, et al. Follow-up analysis of translocation and dicentric frequencies, measured by FISHchromosome painting in breast cancer patients after partialbody radiotherapy with little bone marrow exposure. Mutat Res 1999;446:103–109. Schmidberger H, Virsik-Koepp P, Rave-Frank M, et al. Reciprocal translocations in patients with testicular seminoma before and after radiotherapy. Int J Radiat Oncol Biol Phys 2001;50:857– 864. Bauchinger M, Schmid E, Braselmann H, et al. Time-effect relationship of chromosome aberrations in peripheral lymphocytes after radiation therapy for seminoma. Mutat Res 1989; 211:265–272. Buckton KE, Brown WM, Smith PG. Lymphocyte survival in men treated with x-rays for ankylosing spondylitis. Nature 1967;214:470 – 473. Trepel F. Number and distribution of lymphocytes in man: A critical analysis. Klin Wochensch 1974;52:511–515. Wuestermann PR, Cronkite EP. Physiological and pathophysiological aspects of the immune system contributing to a biomathematical model of lymphocytes. Stem Cells 1995;13: 268 –275. Clave E, Socie G, Cosset JM, et al. Multicolor flow cytometry analysis of blood cell subsets in patients given total body irradiation before bone marrow transplantation. Int J Radiat Oncol Biol Phys 1995;33:881– 886. Svensson C, Petrini B. Changes of gamma/delta T cells in blood after radiation therapy for prostatic cancer. Cancer Lett 1993;72:175–178.
doi:10.1016/j.ijrobp.2005.03.056
CLINICAL INVESTIGATION
Normal Tissues
TIME-COURSE OF RADIATION-INDUCED CHROMOSOMAL ABERRATIONS IN TUMOR PATIENTS AFTER RADIOTHERAPY IRENE MÜLLER,* HANS GEINITZ, M.D.,† HERBERT BRASELMANN,* ADOLF BAUMGARTNER, PH.D.,* ANNETTE FASAN,* REINHARD THAMM,† MICHAEL MOLLS, M.D.,† VIKTOR MEINEKE, M.D.,‡ AND HORST ZITZELSBERGER, M.D.* *GSF-National Research Center for Environment and Health, GmBH, Institute of Molecular Radiation Biology, Neuherberg, Germany; †Department of Radiation Oncology, Klinikum Rechts der Isar, Technical Univeristy, Munich, Germany; ‡Institute of Radiobiology, German Armed Forces, Munich, Germany Purpose: Radiation-induced chromosome aberrations are routinely used in biologic dosimetry to monitor radiation exposure. Translocations are considered stable aberrations with time after exposure. This study was performed to determine the temporal persistence of radiation-induced translocations during a 36-month period in therapeutically irradiated testicular seminoma patients who underwent partial body exposure (>10% of bone marrow). Methods and Materials: Chromosome analyses were carried out in peripheral lymphocytes of 11 patients with testicular seminoma (n ⴝ 9), germinoma (n ⴝ 1), or follicular non-Hodgkin’s lymphoma (n ⴝ 1). All patients received radiotherapy with photons from a linear accelerator; in 1 case, additional electron beams were used. Doses ranged from 26 Gy (seminoma) to 45 Gy (non-Hodgkin’s lymphoma). None of the patients received chemotherapy. From each patient, blood samples were taken during the 36 months after irradiation at defined points. Chromosomal aberrations were scored after fluorescence in situ hybridization painting of chromosomes 1, 4, and 12 in combination with a pancentromeric probe. Results: For 9 patients (7 with testicular seminoma, 1 with germinoma, and 1 with non-Hodgkin’s lymphoma), a significant temporal decline of translocations, with a mean decline rate of 4.4% ⴞ 0.4% monthly, could be detected. Two testicular seminoma patients showed no temporal decline of aberration frequencies. Conclusion: Most partial body irradiated patients (9 of 11) showed a significant temporal decline of translocation frequencies during a 36-month period. Thus, reciprocal translocations after partial body irradiation cannot be regarded as stable over time. The temporal decline of aberration frequencies has to be taken into account for retrospective dose estimations. © 2005 Elsevier Inc. Radiation induced chromosome aberrations, Translocations, Fluorescence in situ hybridization, Temporal decline, Biodosimetry.
INTRODUCTION Radiation-induced chromosomal aberrations can be detected and quantified in peripheral lymphocytes many years after exposure to ionizing irradiation using fluorescence in situ hybridization (FISH) with whole chromosome painting probes (for review, see Bauchinger [1] and Natarajan and Boei [2]). This so-called FISH-painting technique is usually used for retrospective dose estimations and mechanistic investigations on chromosome aberration formation after radiation exposure. For these purposes, a partial genome analysis using combinations of whole chromosome probes for three chromosomes in one or more colors is usually
performed. Recently, techniques for whole genome analyses of radiation-induced chromosome aberrations have also become available (3– 6). However, they are far too time consuming for routine analysis. In biodosimetric applications, translocations occurring in peripheral lymphocytes are considered stable markers of radiation exposure, assuming that they do not decline with time after exposure. This is an important prerequisite for dose reconstructions of past exposures. However, no extensive data have demonstrated the persistence of radiation-induced translocations with time after exposure. In the studies performed so far that have investigated animals (7, 8), victims of accidental irradiation (9 –12), or in vitro irradiated cells (13, 14), contradicting
Reprint requests to: Horst Zitzelsberger, Ph.D., GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Institute of Molecular Radiobiology, Ingolstädter Landstr. 1, Neuherberg D-85764 Germany. Tel: (⫹49) 89-318-73-421; Fax: (⫹49) 89318-72-873; E-mail: [email protected] Supported financially by the Federal Office of Defense Tech-
nology and Procurement, Grant E/B41G/Z0531/Z5803. H. Braselmann and H. Geinitz contributed equally to this study. Acknowledgments—The skillful technical assistance of S. Schröferl and E. Konhäuser is gratefully acknowledged. Received Jan 27, 2005, and in revised form Mar 24, 2005. Accepted for publication Mar 24, 2005. 1214
Temporal decline of radiation-induced translocations
results have been obtained. In another study, the examined cohort of therapeutically irradiated patients was too heterogeneous to receive clear results on the temporal stability of translocations (15). Some of these studies have shown that the translocation frequencies are stable over time (9, 11, 15), and others detected a decline in translocation frequencies (7, 12, 14). The aim of the present study was to evaluate the time course of translocation frequencies in peripheral blood lymphocytes after therapeutic partial body irradiation. We present a data set of patients who received pure radiotherapy (RT) either as the sole treatment for their malignancy or as adjuvant treatment. Translocations were prospectively evaluated before treatment and at defined points up to 36 months after treatment. Decline curves could be calculated for each individual patient.
● I. MÜLLER et al.
1215
approved this study. All patients provided written informed consent before participating.
Cell culture Blood samples were obtained between 1999 and 2003. For each sample, whole blood cultures were set up with heparinized peripheral blood. We added 0.5 mL of blood to each 4.5 mL of Roswell Park Memorial Institute medium 1640 plus Glutamax (Gibco) supplemented with 15% fetal calf serum, 2.5% phytohemagglutinin, antibiotics, and bromodeoxyuridine (at a final concentration of 9.6 ⫻ 10⫺6 g/mL). Cultures were incubated for 45 h at 37°C under 5% carbon dioxide-containing atmosphere followed by a Colcemid treatment (0.1 g/mL) for 3 h under the same conditions. Subsequently, chromosome preparation was performed according to standard procedures (17). The slides were stored at ⫺20°C under nitrogen atmosphere until use.
Fluorescence in situ hybridization METHODS AND MATERIALS Patients Chromosome analyses were performed in 11 male cancer patients who exclusively received RT either as the sole treatment or as adjuvant treatment. No concomitant or sequential chemotherapy was applied. From each patient, at least 10% of the blood cellproducing bone marrow had been irradiated during therapy. Moreover, the survival of each patient was assumed to be ⬎2 years. None of the patients had received any RT or chemotherapy before their current treatment or during follow-up. Nine patients (age 32– 65 years at the beginning of the study) received adjuvant RT after one-sided inguinal orchiectomy for Stage I testicular seminoma. Treatment was given by ventrodorsal opposed fields to a total dose of 26 Gy in 2-Gy fractions, 5 times weekly. The fields extended from the upper border of the 11th thoracic vertebra to the lower border of the 4th lumbar vertebra. Laterally, the fields extended to the edges of the transverse spinal processes. Approximately 12% of the active bone marrow was included in the radiation fields (16). One patient (aged 22 years) received RT for germinoma in the pinealis region. The whole craniospinal axis was treated to a dose of 24 Gy in 1.6-Gy fractions, 5 times weekly. The neurocranium and upper cervical spine were irradiated using two lateral opposed fields, and the remaining spine, including the third sacral vertebra, was treated by two adjoining dorsal fixed fields. After treatment of the craniospinal axis, a boost to the tumor region was applied with a dose of 16 Gy in 1.6-Gy fractions. Approximately 42% of the active bone marrow was included in the radiation fields in this patient (16). One patient (aged 46 years) received definitive RT for follicular non-Hodgkin’s lymphoma Stage Ia of the right inguinal lymph nodes. The whole abdomen and pelvis (“abdominal bath”) were treated with a dose of 25.5. Gy in 1.5-Gy fractions, 5 times weekly. Treatment was done with two ventrodorsal opposed fields. After 12 Gy, the kidneys and part of the liver were shielded. The right groin was treated with electrons to a dose of 45 Gy and the left groin to a dose of 25.2 Gy, both in 1.8-Gy fractions, 5 times weekly. Approximately 50% of the active bone marrow was included in the radiation fields in this patient (16). For each patient, blood samples were taken before RT, immediately after RT, and 2, 5, 8, 12, 18, 24, 30, and 36 months after RT. The ethics committee of the Technical University Munich
Whole chromosome painting probes for chromosomes 1, 4, and 12 were used in combination with a pancentromeric DNA probe. Plasmid DNA of chromosome-specific Hind III pBS libraries of chromosomes 1, 4, and 12 (18) was isolated, as described previously (19). Plasmid DNA samples were labeled with either biotin (chromosomes 1 and 4) or digoxigenin (chromosomes 4 and 12) using nick-translation, as described by Weier et al. (20). A pancentromeric probe was generated and biotinylated by polymerase chain reaction according to Bauchinger et al. (21). After hybridization for 24 – 48 h, bound biotin-labeled probes for chromosomes 1 and 4 and the biotinylated pancentromeric probe were detected with fluorescein isothiocyanate (FITC)streptavidin conjugate (Dianova, Hamburg, Germany) and bound digoxigenin-labeled whole chromosome painting probes for chromosomes 4 and 12 with Cy3-labeled antibodies (Dianova, Hamburg, Germany). Counterstaining was performed with 4=,6diamidinophenylindol (DAPI) in antifade solution (Vecta Shield, Linaris GmbH, Wertheim-Bettingen, Germany) at a concentration of 1 g/mL. To perform chromosome analyses only in the first division metaphases, FISH painting was combined with fluorescence plus Giemsa staining, as described by Kulka et al. (22).
Chromosome analysis After FISH painting, chromosome 1 appeared in green (FITC), chromosome 4 in orange (combination of FITC and Cy3), and chromosome 12 in red (Cy3). The centromeres were additionally stained in green (FITC). Because the FISH signals for the centromeric regions are very bright, they were also visible on chromosomes 1 and 4. Counterstaining of all chromosomes appeared in blue (DAPI). The PAINT (Protocol for Aberration Identification and Nomenclature Terminology) nomenclature system (23), as well as the S&S nomenclature system (24 –26) was applied to describe the observed painting patterns. All images of metaphases exhibiting aberrations of the painted chromosomes were acquired and stored using the ISIS image analysis system (Metasystems, Altlussheim, Germany). For an analysis of the time course of aberrations, reciprocal translocations t(Ab) ⫹ t(Ba) and one-way patterns t(Ba) ⫹ ace(b), t(Ab), and t(Ba) were considered. This corresponds to 2B and 2B I-III according to the S&S nomenclature system. In addition, dicentric chromosomes dic(BA) ⫹ ace(ba), dic(BA) ⫹ ace(b),
Volume 63, Number 4, 2005
Fig. 1. Translocation rates in 11 therapeutically irradiated patients immediately and 2 months after radiotherapy. Error bars indicate standard error of mean for each patient. Two cases (Patients 4 and 9) had significant differences in translocation rates between two time points (asterisks).
1433 (21/9) 555 (14/14)* 1669 (23/18) 1062 (20/15) 1115 (20/3) 833 (41/31) 773 (21/9) NA 1038 (20/11) 1259 (21/14) 355 (12/4)* 1033 (24/14) 442 (11/9)* 1027 (20/27) 661 (22/20) 2011 (22/17) 428 (21/17) NA 585 (20/22) 869 (20/15) 749 (9/19)* 999 (38/19) 1767 (19/18)* 439 (12/7)* 984 (21/10) 661 (24/17) 1181 (21/15) 1151 (20/15) 573 (20/17) 445 (24/22) 514 (8/17)* 886 (20/9) 560 (26/11)
30 mo after RT 24 mo after RT
ace(ba), and dic(BA), corresponding to 2A and 2A IV-VI according to the S&S nomenclature, were taken into account and could be distinguished using the pancentromeric FISH signals.
Statistical analysis Abbreviations: Pt. no. ⫽ patient number; RT ⫽ radiotherapy; NA ⫽ no samples available. * Scoring criteria could not be completely fullfilled owing to limited numbers of slides.
666 (22/18) 480 (22/16) 694 (20/7) 350 (27/20) 1253 (23/15) 442 (15/22)* 411 (21/23) 297 (22/21) 1441 (20/18) 587 (20/11) 504 (39/24) 1918 (0/0) 2027 (2/0) 2623 (0/1) 2107 (1/0) 2139 (0/0) 2253 (0/0) 2051 (0/0) 1960 (0/0) 2106 (1/1) 2064 (1/0) 2300 (1/0) 2 4 5 6 7 8 9 10 11 13 14
376 (20/12) 429 (20/17) 601 (30/24) 191 (24/18) 1023 (33/19) 691 (20/28) 645 (23/19) 770 (37/44) 878 (24/14) 627 (30/22) 285 (69/43)
481 (23/19) 218 (20/25) 574 (37/25) 196 (39/25) 1562 (36/21) 640 (20/25) 257 (21/22) 522 (33/31) 854 (21/10) 378 (23/17) 210 (34/41)
808 (20/17) 298 (20/16) 341 (20/15) 242 (26/20) 781 (38/12) 598 (32/30) 601 (20/29) 466 (32/11) 1029 (20/20) 398 (20/10) 298 (23/31)
735 (20/11) 454 (21/16) 900 (32/32) 612 (38/40) 1889 (32/35) 1430 (42/31) 1002 (26/21) 654 (30/11) 1649 (23/37) 1557 (20/28) 898 (28/27)
18 mo after RT 12 mo after RT 8 mo after RT 5 mo after RT 8 wk after RT After RT Before RT Pt. no.
Cells scored (translocations/dicentrics) (n)
Table 1. Number of translocations and cells scored in 11 irradiated patients at different points
● Biology ● Physics
1421 (20/10) 652 (20/11) 1569 (20/8) 568 (13/6)* 1385 (11/7)* 655 (17/9)* 1303 (26/20) 1710 (24/11) 703 (20/13) 2042 (14/7) 2097 (30/19)
I. J. Radiation Oncology
36 mo after RT
1216
Calculations were performed using an exponential decline model, which was fitted by logarithmic-linear Poisson regression (27). For each patient, a mean infinitesimal decline rate, k (also known as time constant or hazard rate, percentage per month), and mean half-life, h [⫽ ln (2)/k] for translocations and dicentrics, were calculated using follow-up times of between 2 and 36 months after irradiation. Statistical analysis was performed using analysis of deviance (28) with a likelihood-ratio test for intercomparison of decline rates and chi-square goodness-of-fit test for investigation of deviations from the regression model.
RESULTS A total of 109,000 metaphases were scored from 11 therapeutically irradiated patients at 10 different times before and after RT. The criteria for translocation scoring have been established; either 2000 cells or 20 translocations (reciprocal or one-way) had to be scored for each blood sample to obtain sufficient results for additional statistical interpretation (Table 1). The time course of the aberrations was calculated for simple reciprocal and one-way translocations (2B and 2B I-III) and dicentrics with and without acentric fragment, as well as solitary acentric fragments (2A and 2A IV-VI). The initial translocation frequencies were plotted immediately after RT and, for comparison, 8 weeks after RT (Fig. 1). Translocation frequencies immediately after RT were greatest in the 2 patients treated with the highest radiation fields (Patient 6, craniospinal axis and Patient 14, abdominal bath; Fig. 1). Taking all follow-up stages between 8 weeks and 36 months after irradiation into account,
Temporal decline of radiation-induced translocations
Fig. 2. Calculated decline rates for translocations and dicentrics in 9 irradiated patients (7 with testicular seminoma, 1 with germinoma [Patient 6], and 1 with non-Hodgkin’s lymphoma [Patient 14]). *Decline rates for translocations and dicentrics of victim of Estonia radiation accident (whole body exposure, 12). Error bars indicate standard error for each decline rate.
we obtained average decline rates for translocations and dicentrics (Fig. 2). The parameters and statistics from the calculation (i.e., decline rates, extrapolated initial values, chi-square goodness-of-fit test, and half-life periods) are shown in Table 2. For Patients 8 and 11, both treated for testicular seminoma, no temporal decline for translocations could be observed for the complete period (Fig. 3b). In these patients, the initial translocation rates did not differ from the final rates, although a decrease in translocations rates was noted after 12 and 18 months, followed by an increase to the initial levels later. For the other 9 patients, a significant temporal decline
● I. MÜLLER et al.
1217
became apparent for translocations, as well as for dicentrics (Fig. 2). The mean infinitesimal decline rate for translocations in these patients was 4.4%/mo (range, 2.4 – 6.9%). This complies with a half-life of 15.6 months (95% confidence interval, 13.3–18.9). No significant differences in the decline rates of these 9 patients could be detected (likelihood-ratio test, p ⬎ 0.05). An example (Patient 6) of a temporal decline in translocation rates is demonstrated in Fig. 3a. Patients 7, 8, 9, 13, and 14 had significant deviations from a uniform time course (chi-square goodnessof-fit test, p ⬍ 0.05). For Patient 7, the rate of translocations 5 months after irradiation was greater than average. Patients 9 and 13 showed irregular deviations at different times, and Patient 14 exhibited a fast decline within the first year and after 30 months. In Patient 8, reduced rates of translocations were detected during the second year, but the final rates of translocations corresponded to the initial rates.
DISCUSSION The aim of this study was to investigate the time course of translocation frequencies after partial body exposures using FISH painting. Because FISH painting for translocation scoring has been regarded as the gold standard endpoint for radiation biologic dosimetry (29), it appears to be essential to determine the persistence of radiation-induced translocations over time. To date, some studies of whole body exposures after radiation accidents have reported that radiation-induced translocations persist with time (9, 11, 15), although other studies have demonstrated results with a significant decline of translocation frequencies (7, 12, 14). However, in several of these studies, either the initial translocation rates after irradiation were not available or the radiation conditions of the individuals were very inhomo-
Table 2. Statistical analysis (logarithmic-linear Poisson regression) of translocation rates from 8 weeks to 36 months in 11 irradiated patients
Pt. no. 2 4 5 6 7 8 9 10 11 13 14 Mean, without patients 8 and 11
Age (y) 43 65 44 22 32 34 34 39 36 35 46
Malignancy Seminoma Seminoma Seminoma Germinoma Seminoma Seminoma Seminoma Seminoma Seminoma Seminoma NHL
Extrapolated initial values (per 100 cells)
Mean infinitesimal* decline rate k (% mo)
Mean half-life (mo)
Chi-square goodnessof-fit test
3.11 6.75 5.86 15.32 2.75 3.11 4.70 8.11 1.59 4.57 10.44
3.07 ⫾ 1.1 3.38 ⫾ 0.9† 4.92 ⫾ 0.6‡ 6.92 ⫾ 1.1‡ 3.09 ⫾ 1.4‡ ⫺0.06 ⫾ 1.4 2.43 ⫾ 1.1§ 4.67 ⫾ 0.7‡ ⫺0.82 ⫾ 0.8 5.14 ⫾ 1.5‡ 5.43 ⫾ 1.3‡ 4.43 ⫾ 0.39‡
22.6 20.5 14.1 10.0 22.4 — 28.5 14.8 — 13.5 12.8 15.6
12.41 7.26 5.44 11.07 23.04‡ 25.66‡ 12.48§ 4.98 7.14‡ 21.60‡ 26.12‡ 156.5‡
†
Abbreviations: Pt. no. ⫽ patient number; df ⫽ degrees of freedom; NHL ⫽ non-Hodgkin’s lymphoma. * In short intervals ⌬t (⬍1 mo), translocation frequency decreases by k⌬t%. † p ⬍ 0.01. ‡ p ⬍ 0.005. § p ⬍ 0.05.
df 6 6 6 6 6 6 5 5 6 6 6
1218
I. J. Radiation Oncology
● Biology ● Physics
Fig. 3. Time course of radiation-induced translocations in 3 patients. (a) Patient 6 showing decline of translocation frequencies during 36-month period. (b) Patients 8 and 11 showing final unchanged translocation rates after 36 months. Error bars indicate standard error of mean for each translocation rate.
geneous (15), making clear interpretation of the data difficult and possibly explaining the reported discrepancies. Our investigations of therapeutically irradiated patients represent the first study of a largely homogeneously exposed cohort (9 seminoma patients) that included regular blood sampling for each patient to generate decline curves and examine the time course of translocation frequencies after partial body exposure. Published data from therapeutically irradiated patients are also rare. Only a very few studies of the time course of translocation frequencies from partial body irradiated patients have been published (30 – 32). Gebhart et al. (30) reported on a decline of radiationinduced translocations in an inhomogeneous group of patients after several doses and treatment volumes. In another, more homogeneous, cohort of breast cancer patients, the results were not meaningful owing to the small portion of bone marrow in the radiation field (⬍5%), resulting in a nonstatistically significant tendency of a temporal decline in
Volume 63, Number 4, 2005
translocation rates (31). The third study demonstrated a temporal decline in testicular seminoma patients; however, no individual time courses of translocation frequencies at defined times after RT for each patient, and thus no decline curves or statistically evaluated decline rates of translocation frequencies, could be established (32). In our study, individual time courses of translocation frequencies from a largely homogeneously irradiated cohort of patients (9 with testicular seminoma and comparable irradiation conditions, 1 with germinoma, and 1 with nonHodgkin’s lymphoma) could be demonstrated, including the initial translocation rates. As demonstrated in Fig. 2 and exemplary in Fig. 3, only in 2 exposed patients did the translocations persist for a 36-month period, and in 9 exposed patients, a clear decline became apparent, with a mean half-life of 15.6 months. Moreover, the observed dicentric and translocation rates were not significantly different for all patients in our cohort. A reference case from the Estonia accident (12) showed a significantly lower decline rate for translocations than for dicentrics (Fig. 2). This disagreement was probably a result of the different irradiation conditions, which consisted of whole body exposure at a lower dose for the Estonia case and of partial body exposure at high doses for our patient cohort. For a comparison of the half-lives observed in our patient cohort, we reviewed a previous study on dicentrics that had been scored after fluorescence plus Giemsa staining in peripheral lymphocytes from therapeutically irradiated testicular seminoma patients (33). In that study, an initial half-life of about 0.4 years was calculated for dicentrics (33). The unexpected steep decline of translocation rates, with a mean half-life of 15.6 months (Table 2), in the present patient cohort might have resulted from the partial body exposure with high doses of ionizing radiation. Because the half-life of peripheral lymphocytes is limited and a mean life span of 1574 days was estimated (34), cells exhibiting both aberration types, translocations and dicentrics, decline. In contrast, the repopulation of peripheral lymphocytes carrying translocations should be not affected, although dicentrics will disturb cell division of precursor cells and thus the repopulation of lymphocytes. The observation of a decline of peripheral lymphocytes carrying translocations in our patient cohort could be partly attributed to additional lethal aberrations (other than the scored reciprocal translocations) that were present in the same cell. It might, therefore, be argued that translocation scoring in so-called stable cells (i.e., cells that contain only transmissible aberrations during cell cycle) would have resulted in long-term persistence of translocation rates, as demonstrated by Lindholm and Edwards (11). Such “stable” cells have been characterized by also scoring the unpainted part of the genome for the presence of unstable aberrations. This approach is difficult to perform because undetectable complex aberrations that are not transmissible during the cell cycle might also be present in the unpainted part in addition to the dicentrics, rings, and acentrics considered by Lindholm and Edwards (11). We have tried such an analysis in selected blood samples and
Temporal decline of radiation-induced translocations
failed because almost every cell with a translocation carried additional unstable aberrations in the unpainted chromosomes. Additionally, it seems not to be justified for statistical reasons to perform translocation analyses only in “stable” cells, because the same scoring criteria must then be applied to metaphases without translocations in the painted part of the genome. Such a scoring procedure appears not to be feasible if high cell numbers of many individuals must be scored. Moreover, it is evident that not only simple dicentrics in the unpainted part of the genome renders a cell “unstable,” but also complex aberrations may cause a nontransmissible cell during a cell cycle that is not scored in the counterstained part in a FISH-painted metaphase. For a demonstration of the time course of translocation and dicentric frequencies, an exponential model was used for each patient. This resulted in individual decline curves and calculated decline rates (Table 2 and Figs. 2 and 3). Patients 6 and 14 showed the greatest aberration frequencies, in accordance with the larger radiation fields and higher doses applied in these patients. Despite these greater aberration frequencies, a significant deviation from the mean decline rate and the mean half-life of translocations of testicular seminoma patients could not be observed. For 2 patients (Patients 8 and 11), final unchanged translocation rates could be observed after 36 months, although reduced rates became apparent in the second year after irradiation followed by increasing translocation frequencies up to the initial level. This phenomenon could be explained by individually different time courses of lymphocyte repopulation or by persisting irradiated lymphocytes that reenter the blood circulation at a certain point after irradiation. A different volume of active bone marrow in the radiation field
● I. MÜLLER et al.
1219
could be excluded as an explanation for these 2 cases exhibiting final unchanged translocation rates, because the number of vertebrae in the radiation fields was identical among the testicular seminoma patients. A possible explanation for this observation is again the presence of individual cell kinetics of repopulating blood cells from stem cells or the circulation of damaged cells between different lymphocyte-containing compartments. According to a model described by Trepel (35) and Wuestermann and Cronkite (36), lymphocytes are believed to recirculate among the central blood compartment, spleen, lymph nodes, and peripheral tissues, such as the thymus and lymphoid tissue of the gut. Another possibility to explain this phenomenon in our study is the finding that different T-lymphocyte clones exhibit a different radiosensitivity (37) and a different recovery after irradiation (38). Assuming that interindividual variations are present, such observations also may influence the time course of aberration-carrying lymphocytes after therapeutic irradiation. CONCLUSION We investigated 11 therapeutically irradiated patients during a 36-month period for radiation-induced translocation frequencies. We observed a significant temporal decline in translocation frequencies in 9 patients. In 2 patients, the translocation frequencies persisted within this period. The decline of radiation-induced translocations with time might have been a result of the partial body exposure in the high-dose range. The calculated decline rates have an impact on retrospective dose estimations, especially in the high-dose range of partial body irradiation.
REFERENCES 1. Bauchinger M. Retrospective dose reconstruction of human radiation exposure by FISH/chromosome painting. Mutat Res 1998;404:89 –96. 2. Natarajan AT, Boei JJ. Formation of chromosome aberrations: Insights from FISH. Mutat Res 2003;544:299 –304. 3. Schrock E, du Manoir S, Veldman T, et al. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494 – 497. 4. Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12:368 –375. 5. Greulich KM, Kreja L, Heinze B, et al. Rapid detection of radiation-induced chromosomal aberrations in lymphocytes and hematopoietic progenitor cells by mFISH. Mutat Res 2000;452:73– 81. 6. Loucas BD, Cornforth MN. Complex chromosome exchanges induced by gamma rays in human lymphocytes: An mFISH study. Radiat Res 2001;155:660 – 671. 7. Tucker JD, Breneman JW, Briner JF, et al. Persistence of radiation-induced translocations in rat peripheral blood determined by chromosome painting. Environ Mol Mutagen 1997; 30:264 –272. 8. Tucker JD, Marples B, Ramsey MJ, et al. Persistence of chromosome aberrations in mice acutely exposed to 56Fe⫹26 ions. Radiat Res 2004;161:648 – 655. 9. Salassidis K, Georgiadou-Schumacher V, Braselmann H, et al.
10. 11. 12. 13.
14.
15.
Chromosome painting in highly irradiated Chernobyl victims: A follow-up study to evaluate the stability of symmetrical translocations and the influence of clonal aberrations for retrospective dose estimation. Int J Radiat Biol 1995;68:257–262. Lindholm C, Tekkel M, Veidebaum T, et al. Persistence of translocations after accidental exposure to ionizing radiation. Int J Radiat Biol 1998;74:565–571. Lindholm C, Edwards A. Long-term persistence of translocations in stable lymphocytes from victims of a radiological accident. Int J Radiat Biol 2004;80:559 –566. Bauchinger M, Schmid E, Braselmann H. Time-course of translocation and dicentric frequencies in a radiation accident case. Int J Radiat Biol 2001;77:553–557. Boei JJ, Vermeulen S, Natarajan AT. Detection of chromosomal aberrations by fluorescence in situ hybridization in the first three postirradiation divisions of human lymphocytes. Mutat Res 1996;349:127–135. Matsumoto K, Ramsey MJ, Nelson DO, et al. Persistence of radiation-induced translocations in human peripheral blood determined by chromosome painting. Radiat Res 1998; 149:602– 613. Tawn EJ, Whitehouse CA. Persistence of translocation frequencies in blood lymphocytes following radiotherapy: Implications for retrospective radiation biodosimetry. J Radiol Prot 2003;23:423– 430.
1220
I. J. Radiation Oncology
● Biology ● Physics
16. Ellis RE. The distribution of active bone marrow in the adult. Phys Med Biol 1961;5:255–258. 17. Huber R, Kulka U, Lorch T, et al. Technical report: Application of the Metafer2 fluorescence scanning system for the analysis of radiation-induced chromosome aberrations measured by FISH-chromosome painting. Mutat Res 2001;492: 51–57. 18. Pinkel D, Landegent J, Collins C, et al. Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 1988;85:9138 –9142. 19. Schmid E, Zitzelsberger H, Braselmann H, et al. Radiationinduced chromosome aberrations analysed by fluorescence in situ hybridization with a triple combination of composite whole chromosome-specific DNA probes. Int J Radiat Biol 1992;62:673– 678. 20. Weier HU, Lucas JN, Poggensee M, et al. Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations. Chromosoma 1991;100:371–376. 21. Bauchinger M, Schmid E, Zitzelsberger H, et al. Radiationinduced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe. Int J Radiat Biol 1993;64:179 –184. 22. Kulka U, Huber R, Muller P, et al. Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes. Int J Radiat Biol 1995;68: 25–27. 23. Tucker JD, Morgan WF, Awa AA, et al. PAINT: A proposed nomenclature for structural aberrations detected by whole chromosome painting. Mutat Res 1995;347:21–24. 24. Savage JR, Simpson P. On the scoring of FISH-“painted” chromosome-type exchange aberrations. Mutat Res 1994;307: 345–353. 25. Savage JR, Simpson PJ. FISH “painting” patterns resulting from complex exchanges. Mutat Res 1994;312:51– 60. 26. Simpson PJ, Savage JR. Dose-response curves for simple and complex chromosome aberrations induced by X-rays and de-
Volume 63, Number 4, 2005
27. 28. 29. 30. 31.
32.
33.
34. 35. 36.
37.
38.
tected using fluorescence in situ hybridization. Int J Radiat Biol 1996;69:429 – 436. Frome EL. The analysis of rates using Poisson regression models. Biometrics 1983;39:665– 674. Nelder JA, Wedderburn RWM. Generalized linear models. J R Statist Soc A 1972;135:370 –384. Tucker JD. Sensitivity, specificity, and persistence of chromosome translocations for radiation biodosimetry. Mil Med 2002; 167:8 –9. Gebhart E, Neubauer S, Schmitt G, et al. Use of a three-color chromosome in situ suppression technique for the detection of past radiation exposure. Radiat Res 1996;145:47–52. Huber R, Braselmann H, Kulka U, et al. Follow-up analysis of translocation and dicentric frequencies, measured by FISHchromosome painting in breast cancer patients after partialbody radiotherapy with little bone marrow exposure. Mutat Res 1999;446:103–109. Schmidberger H, Virsik-Koepp P, Rave-Frank M, et al. Reciprocal translocations in patients with testicular seminoma before and after radiotherapy. Int J Radiat Oncol Biol Phys 2001;50:857– 864. Bauchinger M, Schmid E, Braselmann H, et al. Time-effect relationship of chromosome aberrations in peripheral lymphocytes after radiation therapy for seminoma. Mutat Res 1989; 211:265–272. Buckton KE, Brown WM, Smith PG. Lymphocyte survival in men treated with x-rays for ankylosing spondylitis. Nature 1967;214:470 – 473. Trepel F. Number and distribution of lymphocytes in man: A critical analysis. Klin Wochensch 1974;52:511–515. Wuestermann PR, Cronkite EP. Physiological and pathophysiological aspects of the immune system contributing to a biomathematical model of lymphocytes. Stem Cells 1995;13: 268 –275. Clave E, Socie G, Cosset JM, et al. Multicolor flow cytometry analysis of blood cell subsets in patients given total body irradiation before bone marrow transplantation. Int J Radiat Oncol Biol Phys 1995;33:881– 886. Svensson C, Petrini B. Changes of gamma/delta T cells in blood after radiation therapy for prostatic cancer. Cancer Lett 1993;72:175–178.