E. Janet Tawn

Genetic effects of radiotherapy for childhood cancer.

Abstract— Radiation-induced heritable diseases have not been demonstrated in humans and estimates of genetic risks for protection purposes are based on mouse experiments. The most comprehensive epidemiologic study is of the Japanese atomic bomb survivors and their children, which found little evidence for inherited defects attributable to parental radiation. Studies of workers exposed to occupational radiation or of populations exposed to environmental radiation appear too small and exposures too low to convincingly detect inherited genetic damage. In contrast, survivors of childhood cancer form the largest group of people exposed to high doses of ionizing radiation before reproduction and offer unique advantages for studying trans-generation effects. A wide range of gonadal doses are possible, several comparison groups are readily available (including siblings), and there is a strong willingness among cancer survivors to participate in health studies. Cancer patients also have detailed medical records that facilitate both the accurate estimation of gonadal doses and the assessment of potentially confounding factors, such as intercurrent illness, personal and family medical histories, lifestyle characteristics such as tobacco use, and circumstances at delivery. An international study is nearing completion of over 25,000 survivors of childhood cancer in the United States and Denmark who gave birth to or fathered over 6,000 children. Doses to gonads are being reconstructed from radiotherapy records with 46% over 100 mSv and 16% over 1,000 mSv. Adverse pregnancy outcomes being evaluated include major congenital malformations, cytogenetic abnormalities, stillbirths, miscarriages, neonatal deaths, total deaths, leukemia and childhood cancers, altered sex ratio, and birth weight. The main analyses are based on dose-response evaluations. Blood studies of trios (cancer survivor, spouse or partner and offspring) have been initiated to evaluate mechanistic evidence for the transmission of any radiation-induced genetic damage such as minisatellite mutations. Markers of cancer susceptibility such as chromosomal radiosensitivity and genotype profile will also be examined. In the United States series to date, 4,214 children were born to cancer survivors among whom 157 (3.7%) genetic diseases were reported in contrast to 95 (4.1%) reported conditions among 2,339 children born to sibling controls. In the Denmark series the comparable figures were 82 (6.1%) birth defects among 1,345 children of cancer survivors and 211 (5.0%) among 4,225 children of sibling controls. Coupled with prior studies, these preliminary findings, if sustained by ongoing dose-response analyses, provide reassurance that cancer treatments including radiotherapy do not carry much if any risk for inherited genetic disease in offspring conceived after exposure.

Chromosomal Abnormalities among Offspring of Childhood-Cancer Survivors in Denmark: A Population-Based Study

Ionizing radiation and many cancer drugs have the potential to produce germ-cell mutations that might lead to genetic disease in the next generation. In a population-based study, we identified, from records in the Danish Cancer Registry, 4,676 children treated for cancer. Their 6,441 siblings provided a comparison cohort. The results of a search of the Central Population Register identified 2,630 live-born offspring of the survivors and 5,504 live-born offspring of their siblings. The occurrence of abnormal karyotypes diagnosed in these offspring and also in any pregnancies terminated following prenatal diagnosis of a chromosome abnormality was determined from the Danish Cytogenetic Registry. After exclusion of hereditary cases and inclusion of the prenatal cases, after correction for expected viability, the adjusted proportion of live-born children in survivor families with abnormal karyotypes (5.5/2,631.5 [0.21%]) was the same as that among the comparison sibling families (11.8/5,505.8 [0.21%]). There were no significant differences in the occurrence of Down syndrome (relative risk [RR]=1.07; 95% CI 0.16-5.47) or Turner syndrome (RR=1.32; 95% CI 0.17-7.96) among the children of cancer survivors, compared with the children of their siblings. These reassuring results are of importance to the survivors, to their families, and to genetic counselors.

FISH Chromosome Aberration Analysis on Retired Radiation Workers from the Sellafield Nuclear Facility

Abstract Tawn, E. J., Whitehouse, C. A. and Tarone, R. E. FISH Chromosome Aberration Analysis on Retired Radiation Workers from the Sellafield Nuclear Facility. Radiat. Res. 162, 249–256 (2004). Chromosome analysis using fluorescence in situ hybridization was undertaken on 294 retired workers from the British Nuclear Fuels plc facility at Sellafield, 95 with external occupational exposure <50 mSv, 108 with 50–499 mSv, and 91 with >500 mSv. In univariate analyses, external dose (P < 10−5) and age (P = 0.0075) were significantly associated with translocation frequency, but no effect was found for smoking status. In a multivariate analysis with age and external dose as continuous variables, the slopes were 0.017 ± 0.0075 × 10−2 translocations per cell per year for age (P = 0.024) and 1.11 ± 0.190 × 10−2 translocations per cell per sievert for external dose (P < 10−5). The dose response for translocation induction for occupational workers is similar to the linear component of in vitro dose–response curves, thus supporting the use of translocation frequency for retrospective biological dosimetry in situations of chronic low-dose exposure occurring over many years. The dose response obtained in this study is lower than the linear component of the dose response for stable chromosome aberrations obtained for the Japanese atomic bomb survivors. Thus, if chromosome aberration levels are indicative of cancer risk, this would suggest that low-dose risks derived from the Japanese atomic bomb survivor data will overestimate the risks associated with the occupational exposure encountered by the men in this study.

Chromosome studies in plutonium workers

Chromosome analyses have been performed on peripheral blood lymphocytes from 54 men with estimates of plutonium body burdens in excess of 296 Bq. Both stable and unstable aberrations were scored using a banding technique and breakpoints noted. In discussing the significance of aberration frequencies the relative proportions of the different types of aberration and their distribution have been considered and account has been taken of external radiation exposure. It is suggested that significant depositions of plutonium do cause an increase in chromosome aberrations. The distribution of the breakpoints in the controls showed an excess in chromosomes 7 and 14. The formation and survival of radiation-induced breakpoints was randomly distributed amongst the chromosomes according to length. The distribution of the breakpoints within the chromosomes showed an excess in the centromeres and telomeres. Possible hot spots occurred in some of these regions and also in certain bands of the intermediate regions of the chromosomes.

Frequencies of chromosome aberrations in a control population determined by G banding.

Control data on chromosome aberration frequencies determined by G banding from several studies undertaken in this laboratory have been re-analysed in relation to age and smoking. The combined study group comprised a total of 162 men (90 non-smokers and 72 smokers) with ages ranging from 20 to 72 years. For the group as a whole, significant increases were observed in translocations and all symmetrical exchanges with increasing age and also when smokers were compared with non-smokers.

Sequential chromosome aberration analysis following radiotherapy - no evidence for enhanced genomic instability.

Chromosome analysis of peripheral blood lymphocytes using block staining was performed on 18 cancer patients who had received fractionated radiotherapy doses totalling 35-80 Gy. Samples were obtained from 13 individuals within 1 year of treatment and thereafter approximately annually up to a maximum of eight times (range: three to eight samples per individual). Sampling of the remaining five patients started later. Frequencies of cells with unstable chromosome aberrations showed a steady decline whereas frequencies of cells with just chromatid aberrations and gaps were initially low and remained so. There was no subsequent rise in any aberrant cell type in later years and thus no suggestion that the radiation exposure had induced a persistent or late manifesting state of genomic instability.

The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mothers age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.

Persistence of translocation frequencies in blood lymphocytes following radiotherapy: implications for retrospective radiation biodosimetry

Chromosome aberration analysis using a G-banding technique was performed on peripheral blood lymphocyte cultures from eight individuals over a 5 year period following therapeutic radiation exposure. Samples were placed in three time periods comprising 0-12, 12-36 and 36-60 months post-treatment. The group was heterogeneous with respect to exposure and this resulted in wide differences in initial total translocation yields. Total translocation frequencies declined in seven of the eight cases, reaching significance in four cases. This decline was attributed to a decrease in cells, which in addition to translocations, also contained aberrations such as dicentrics which resulted in them being unstable. In all eight cases, when only stable cells were considered, no significant differences were observed in translocation frequencies between the different time periods post-treatment. Thus, although the frequency of translocations in stable cells is persistent over time, extrapolating to total initial yield, and using this to equate to dose, is not possible in cases where the exposure has been high and non-homogeneous. In practice, retrospective biological dosimetry is more often required in cases of historical, usually protracted, exposures which will have been essentially uniform and not of a sufficiently high dose for many cells to have acquired more than one aberration. In such cases the frequency of translocations observed some years after the exposure can be assumed to reflect induced frequencies and be used for dose estimation.

Chromosome intra- and inter-changes determined by G-banding in radiation workers with in vivo exposure to plutonium

Suggestions that exposure to intakes of alpha-emitting radionuclides such as plutonium could result in a specific profile of chromosome damage distinguishable from that of low LET irradiation have led to the re-analysis of the different types of chromosome aberrations in peripheral blood lymphocytes determined by G-banding in a group of 20 plutonium workers from the British Nuclear Fuels plc facility at Sellafield, UK. Comparisons were made with a group of workers with negligible plutonium intakes but similar external gamma doses and with an unexposed control group. Examination of simple translocation frequencies in the three groups indicated a significant difference (P = 0.033), with the higher frequency in the plutonium workers indicating that exposure from plutonium was contributing to the aberration yield. Slightly raised frequencies of both intra-chromosomal and complex aberrations were observed in the plutonium workers in comparison with the comparable external exposure group and the control group but the difference did not reach significance at the P = 0.05 level and there was no variation in the relative frequencies of the different aberration types between the three groups. There was, therefore, no firm indication from this study that either intra-chromosomal or complex aberrations could be used as a specific marker of high LET exposure in workers with historical intakes of plutonium.

Mitochondrial DNA mutations in individuals occupationally exposed to ionizing radiation.

Abstract Wilding, C. S., Cadwell, K., Tawn, E. J., Relton, C. L., Taylor, G. A., Chinnery, P. F. and Turnbull, D. M. Mitochondrial DNA Mutations in Individuals Occupationally Exposed to Ionizing Radiation. Radiat. Res. 165, 202–207 (2006). Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of >0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 × 10−5 mutations/ nucleotide, and for the controls it was 8.65 × 10−5 mutations/ nucleotide, with a procedural error rate of 2.65 × 10−5 mutations/nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P = 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-dose-rate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation.