Persistence of translocation frequencies in blood lymphocytes following radiotherapy:

implications for retrospective radiation biodosimetry

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2003 J. Radiol. Prot. 23 423

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INSTITUTE OF PHYSICS PUBLISHING JOURNAL OF RADIOLOGICAL PROTECTION

J. Radiol. Prot. 23 (2003) 423–430 PII: S0952-4746(03)69765-X

Persistence of translocation frequencies in bloodlymphocytes following radiotherapy: implications forretrospective radiation biodosimetry

E Janet Tawn1 and Caroline A Whitehouse

Genetics Unit, Westlakes Research Institute, Moor Row, Cumbria CA24 3JY, UK

E-mail: Jan.Tawn@westlakes.ac.uk

Received 13 February 2003, in final form 10 May 2003, accepted forpublication 20 June 2003Published 8 December 2003Online at stacks.iop.org/JRP/23/423

AbstractChromosome aberration analysis using a G-banding technique was performedon peripheral blood lymphocyte cultures from eight individuals over a 5 yearperiod following therapeutic radiation exposure. Samples were placed in threetime periods comprising 0–12, 12–36 and 36–60 months post-treatment. Thegroup was heterogeneous with respect to exposure and this resulted in widedifferences in initial total translocation yields. Total translocation frequenciesdeclined in seven of the eight cases, reaching significance in four cases. Thisdecline was attributed to a decrease in cells, which in addition to translocations,also contained aberrations such as dicentrics which resulted in them beingunstable. In all eight cases, when only stable cells were considered, nosignificant differences were observed in translocation frequencies betweenthe different time periods post-treatment. Thus, although the frequency oftranslocations in stable cells is persistent over time, extrapolating to total initialyield, and using this to equate to dose, is not possible in cases where the exposurehas been high and non-homogeneous. In practice, retrospective biologicaldosimetry is more often required in cases of historical, usually protracted,exposures which will have been essentially uniform and not of a sufficientlyhigh dose for many cells to have acquired more than one aberration. In suchcases the frequency of translocations observed some years after the exposurecan be assumed to reflect induced frequencies and be used for dose estimation.

1. Introduction

The use of translocation frequencies in peripheral blood lymphocytes as a measure of historicaland cumulative radiation dose relies on the assumption that frequencies remain constant over

1 Author to whom any correspondence should be addressed.

0952-4746/03/040423+08$30.00 © 2003 IOP Publishing Ltd Printed in the UK 423

424 E J Tawn and C A Whitehouse

a number of years after exposure. Support for this view comes from studies of the Japaneseatomic bomb survivors (Lucas et al 1992) and former inhabitants of a contaminated buildingin Taiwan (Hsieh et al 2001) in which translocation frequencies have been directly related tophysical dose estimates. In addition, follow-up studies on an individual who had accidentallyincorporated tritiated water (Lloyd et al 1998) and radiation workers accidentally exposed toexternal radiation (Stephan and Pressl 1997) have demonstrated that translocation frequenciessome years later are similar to dicentric frequencies used for traditional dose estimationsobserved at the time of exposure. Such studies also provide evidence that haemopoieticstem cells and peripheral blood lymphocytes have the same radiosensitivity for chromosomedamage. However, a study which started 5 years after the Goiania accident,and extended over 3years (Natarajan et al 1998), observed no overall time trend for the persistence of translocations,with some individuals exhibiting a decline and some an increase in frequency. Furthermore, thetranslocation frequencies were two to three times lower than the initial frequencies of dicentricsseen shortly after the accident. Thus uncertainty still remains regarding the persistenceof translocation frequencies in peripheral blood lymphocytes and the relationship betweenfrequencies shortly after exposure and those determined years later. This inevitably raisesquestions regarding the validity of using translocation frequencies observed some time afterthe exposure to determine the extent of the radiation dose.

As part of a European Commission Concerted Action Programme examining this issue wewere prompted to re-examine historical data on radiotherapy patients who were sampled shortlyafter treatment and again a number of times during the following years. Because a G-bandingtechnique was employed it was possible to distinguish both symmetrical, i.e. translocations,insertions, inversions, and asymmetrical aberrations, i.e. dicentrics, rings, fragments. For thepurpose of this report we are interested in the persistence of translocations and how this maybe influenced by the initial distribution of aberrations between stable and unstable cells.

2. Methods

2.1. Study group

The study population comprised eight individuals who had received radiotherapy treatmentfor various types of cancer. Details of patient data, treatment regimes and age at treatmentare shown in table 1. Disease types comprise Hodgkin’s lymphoma and cancer of the larynx,breast, bladder, ovary, pituitary and nose. The total doses received ranged from 40 to 80 Gydelivered in 20–35 fractions of 2.0–2.5 Gy. With the exception of one individual with cancer ofthe larynx, all were still alive at the end of 1998. Each individual was sampled between threeand five times, the first sample being received within 12 months of completion of radiotherapytreatment and subsequent samples up to 60 months post-treatment.

2.2. Lymphocyte culture

Peripheral blood lymphocytes were cultured for 48 h at 37 ◦C in Eagles medium supplementedwith 15% foetal bovine serum and 2% phytohaemagglutinin. Colcemid was added for thefinal 4 h prior to harvesting at a final concentration of 0.1 µg ml−1. Cells were harvested byexposure to a hypotonic solution of 75 mM KCl followed by fixation with methanol and aceticacid in the ratio of 3:1. Fixed cells were stored at −20 ◦C until required.

Persistence of translocations following radiotherapy 425

Table 1. Individual patient data and treatment regimes.

Total Date Age atradiotherapy Number of Fractionated Fractionation treatment treatment

Patient Sex Malignancy dose (Gy) fractions dose (Gy) time (days) ended end (years)

1 M Hodgkin’s 40 20 2.0 30 19/05/81 202 M Larynx 60 25 2.4 35 06/12/84 423 M Larynx 64 30 2.1 42 28/11/79 564 F Breast∗ 80 35 2.3 47 25/07/85 415 M Bladder 60 24 2.5 24 01/11/85 526 F Ovary 45 20 2.3 28 08/08/84 547 F Pituitary 40 20 2.0 29 06/07/83 448 M Nose 50 20 2.5 31 30/07/82 60

∗ Also had two iridium implants during 04/85 and 06/85.

2.3. Aberration analysis

Chromosome analysis was undertaken on slides G-banded with trypsin to a resolution of400 bands per cell. All observable aberrations were recorded and classed as translocations,inversions, dicentrics, centric rings, interstitial and terminal deletions, excess acentricfragments and complex rearrangements involving three or more breaks in two or morechromosomes.

Between 30 and 100 cells were fully analysed from each sample for each individual.Translocations, inversions and stable complexes were classified as symmetrical aberrationswhile dicentrics, centric rings and unstable complexes were classified as asymmetricalaberrations. Excess fragments, terminal and interstitial deletions were classified separatelyas fragments. Any cell containing an asymmetrical aberration or fragment, regardless ofwhether it also contained a symmetrical aberration, was classed as unstable, conversely a cellcontaining only symmetrical aberrations was classed as stable.

3. Results

The results for each individual case were calculated in three broad time periods since samplingdid not commence at exactly the same time for each individual and was not subsequentlyundertaken on a consistent basis. The three time periods were 0–12 months, 12–36 monthsand 36–60 months post-treatment. The total number of cells analysed per sample for eachcase in each time period is given in table 2. Some cases were sampled more than once ineach time period and numbers of cells analysed have been placed in date order. Frequencies oftranslocations in all cells and for translocations in stable cells only were calculated for combinedsamples from each individual case within each time period,plots of which are shown in figure 1.Stable cells are those that appear karyotypically normal plus those damaged cells where allaberrations are monocentric. The number of stable cells was therefore calculated by deductingthe number of unstable cells from the total number analysed.

Statistical analysis using the likelihood-ratio χ2 test was carried out to compare samplesfrom each individual in the three different time periods. Total frequencies of translocationsdeclined over time in all individuals with the exception of case 5 who demonstrated a slightincrease. This decline in frequency reached statistical significance in cases 3 ( p = 0.011),4 (p = 0.048), 6 (p < 0.001) and 8 (p = 0.037). In contrast, although frequencies oftranslocations in stable cells showed slight fluctuations, no statistical difference between anytime period was observed for any individual. In all cases the total frequency of translocations

426 E J Tawn and C A Whitehouse

Figure 1. Frequencies, ± standard errors, of total translocations in all cells and translocations instable cells for each case: ◦, total translocation frequency; �, translocations in stable cells.

Table 2. Number of cells fully analysed by G-banding in each time period.

Time post-treatment (months)

Case 0–12 12–36 36–60 Total number of samples

1 50 60 34, 49, 46 52 100 — 32, 57 33 49 55 31, 30, 50 54 40, 44 — 90 35 100 50, 60 — 3∗6 33 99 81, 100 47 100 100, 33 100 48 52 100, 100 50 4

∗ Analysis also carried out on 354 genome equivalents analysed by the fluorescence in situhybridisation technique 167 months post-treatment.

had declined by the third time period to a very similar frequency to that for translocations instable cells only.

Case 5, in addition to the two G-banded samples (table 2, figure 1), underwent a furtherchromosome analysis at 167 months post-treatment using the fluorescence in situ hybridisationtechnique (FISH). One thousand metaphase spreads were analysed according to our standardprocedure (Bothwell et al 2000). This equates to 345 genome equivalents or fully analysed G-banded cells. In this particular individual, translocations in unstable cells made no contributionto initial total translocation frequency, and the frequency of translocations of 0.035 ± 0.01 percell in this later sample was not statistically different from those from the previous two samplingtimes.

Persistence of translocations following radiotherapy 427

4. Discussion

The persistence of stable aberrations in peripheral blood lymphocytes following radiationexposure has long been recognised by those studying the after effects of radiotherapy, althoughlittle has been done to quantify the frequencies in relation to the time course following exposure.A G-banding study of a group of survivors of childhood leukaemia (Rubin et al 1986) reportedsimilar overall frequencies of chromosomally aberrant cells at 3.9 and 7.2 years post-exposurebut the initial yield after treatment was not examined. A group of thyroid patients displayedsimilar raised frequencies of translocations 1 week and 3.5 years after treatment with radioactiveiodine (Puerto et al 2000). However, in a study of five breast cancer patients who received highfractionated localised radiotherapy, three showed no decline in dicentrics or translocations overthe subsequent 14 months, whereas a significant decline in both aberration types was seen forthe other two patients (Huber et al 1999). This was attributed to differences in lymphocyteturnover and it was anticipated that a decline would eventually be apparent for all five patients.Similar frequencies of translocations were also observed at 4 and 12 months after treatment ina series of breast cancer patients treated with radiotherapy (Legal et al 2002) but lymphocyteturnover between the two sampling times is unlikely to have had a significant effect in thisstudy.

In cases of partial body exposure the aberrations are confined to that fraction of the cellswhich was irradiated. Thus for any given initial yield the aberrations will be confined to fewercells than for a uniform exposure and there will be more cells containing both dicentrics andtranslocations. Since dicentrics encounter difficulties when passing through cell division, suchcells will be unstable. In vitro simulation experiments (Guerrero-Carbajal et al 1998, Lloyd1998) have led to suggestions that the decline in translocation frequency with time seen insome studies of high partial body exposure (e.g. Schmidberger et al 2001) is the result of lossof unstable cells which also contain translocations. Support for this view comes from a 2 yearfollow-up study on five individuals accidentally exposed to a radioactive source in Estonia(Lindholm et al 1998). Translocation frequencies remained relatively stable in the four caseswith predominantly whole body exposure but for the case with additional partial body exposure,which resulted in hand burns, there was a decline in translocation yield. In a subsequent moreextensive analysis of three of the individuals over a 4 year period (Lindholm et al 2002), itwas suggested that translocation frequencies had declined in all three cases to reach a similarfrequency, despite one individual having received an estimated dose considerably higher thanthe other two and having a higher initial translocation yield.

In vitro FISH studies have shown that asymmetrical aberrations are virtually eliminatedafter 20 cell cycles and more than two-thirds of cells with complex rearrangements have alsodisappeared (Littlefield et al 2000). In contrast, the frequencies of reciprocal translocationsare maintained and show a good correlation with dose. In an accompanying study of a groupof radiotherapy patients using solid stained material, cells with abnormal monocentrics, butno asymmetrical aberrations, remained at similar frequencies over a 17 year period. However,patients with similar average bone marrow doses had variable frequencies. This was attributedto the differing nature of the complex multiple fractionated radiotherapy regimes which wouldhave resulted in different proportions of exposed and non-exposed cells. As a result, althoughthe frequency of induced aberrations may have initially been the same, the distribution ofaberrations among cells will have differed, and the proportion of cells carrying only stableaberrations and thus capable of being maintained in the dividing population will vary amongindividuals. The extent of this difference in aberration distribution resulting from differentregimes of exposure can be seen in the eight individuals presented here. Examination of thefirst time period indicates a wide variation in the contribution of translocations in stable cells to

428 E J Tawn and C A Whitehouse

the total translocation frequency. In all cases, with the exception of case 5, total translocationfrequencies decline with time whereas translocation frequencies in stable cells remain more orless constant and at the last time period account for nearly all of the translocations observed.Case 5 differs from the rest in that the initial translocation frequency relies on no translocationsfrom unstable cells. This explains why the frequency does not change significantly at thesubsequent sampling time and why there was no significant difference between these twofrequencies and that obtained for an additional sample taken after this study was finished. Thus,although the frequency of translocations in stable cells is persistent over time, extrapolatingto initial yield and equating this with dose will be difficult in cases where the exposure hasbeen high and non-homogeneous. However, in practice, retrospective biological dosimetry isnot usually required in such situations since deterministic effects will become apparent shortlyafter exposure. Of more concern is establishing the extent of historical exposures for which noobvious effects have been observed but which might confer increased long-term risks whichneed to be evaluated. Experiments in vitro indicate that for uniform acute exposures restrictingthe analysis to stable cells only results in a marked reduction in overall translocation yieldsonce doses reach 3 Gy (Finnon et al 1999) and for chronic exposure this will be even greater.Similar results were found by Spruill et al (2000) in a lifespan study of mice who had receivedwhole body acute irradiation at doses ranging from 0 to 4 Gy. With doses at and below 2 Gy nodecrease in translocation frequency with time was observed. Furthermore, historical exposuresto external sources requiring dose evaluation tend to be protracted in nature and thus exposure tothe haemopoietic cells will be essentially uniform. In such cases the translocation frequenciessome years after exposure will tend to reflect induced frequencies and can therefore be usedfor dose estimation.

Acknowledgment

We thank Fiona Martin for contributing to the cytogenetic analysis.

Resume

Par la methode de la bande G, on a effectue l’analyse d’aberration chromosomique sur descultures de sang peripherique venant de huit personnes, durant une periode de cinq ans apresleur exposition therapeutique au rayonnement. On a recueilli des echantillons durant troisperiodes de temps, zero a 12 mois, 12 a 36 mois, 36 a 60 mois apres le traitement. L’expositiondu groupe etait heterogene; il en est resulte de larges differences du seuil initial de translocationtotale. Dans sept des huit cas, les frequences de translocation totale declinaient; elles ont etesignificatives dans quatre cas. On a attribue ce declin a la diminution des cellules qui, enplus d’une translocation, contenaient aussi des aberrations, telles que des dicentriques, qui lesrendaient instables. Quand on ne considerait que des cellules stables, dans les huit cas onn’a observe aucune difference significative des frequences de translocation, quelles que soientles periodes de temps apres le traitement. Au total, bien que la frequence des translocationsdans les cellules soit temporellement persistante, il n’est pas possible d’extrapoler jusqu’auseuil initial total, et de l’employer pour parametriser la dose, dans le cas ou l’exposition aete importante et non homogene. Dans la pratique, la dosimetrie biologique retrospective estplus souvent indiquee dans des cas d’expositions historiques, habituellement prolongees, etessentiellement uniformes; elle n’est pas adaptee au cas d’une dose assez elevee pour que denombreuses cellules aient subi plus d’une aberration. Dans de tels cas, on peut supposer quela frequence des translocations observees plusieurs annees apres l’exposition reflete bien lesfrequences induites; on peut l’utiliser pour estimer la dose.

Persistence of translocations following radiotherapy 429

Zusammenfassung

Eine Chromosomen-Aberrationsanalyse unter Verwendung einer G-Banding-Technik wurdean peripheren Blutlymphozytenkulturen von acht Personen uber einen Zeitraum von funfJahren nach einer therapeutischen Strahlenbehandlung durchgefuhrt. Die Proben wurdenin drei Zeitraumen von von 0 bis 12, 12 bis 36 und 36 bis 60 Monaten nach derBehandlung untersucht. Die Gruppe war hinsichtlich der Belastung heterogen—dies fuhrtezu großen Unterschieden bei den ersten gesamten Translokationsergebnissen. Die gesamtenTranslokationshaufigkeiten nahmen in sieben der acht Falle ab, sie erreichten Signifikanzin vier Fallen. Diese Abnahme wurde einer Verringerung der Zellen zugeschrieben, dieneben Translokationen auch Aberrationen enthielten, wie Dicentrics, was dazu fuhrte, dasssie instabil wurden. Als nur stabile Zellen einbezogen wurden, wurden in allen acht Fallenkeine signifikanten Unterschiede in den Translokationshaufigkeiten bei den unterschiedlichenZeitraumen nach der Behandlung beobachtet. Obwohl die Haufigkeit der Translokationenbei stabilen Zellen uber einen Zeitraum anhaltend ist, ist eine Extrapolation auf das gesamteerste Ergebnis und Verwendung dieses Ergebnisses zur Gleichsetzung mit der Dosis nichtmoglich in Fallen, bei denen die Bestrahlung hoch und nicht homogen war. In der Praxis istdie retrospektive biologische Dosimetrie ofter erforderlich in Fallen historischer, gewohnlichverschleppter Bestrahlungen, die im wesentlichen einheitlich waren und bei denen die Dosisfur viele Zellen nicht ausreichend hoch war, um mehr als eine Abweichung aufzuweisen. Indiesen Fallen kann man davon ausgehen, dass die Haufigkeit der Translokationen, die einigeJahre nach der Bestrahlung beobachtet wurde, induzierte Haufigkeiten widerspiegelt und zurDosisabschatzung eingesetzt werden kann.

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