Table 1. Human leukaemogens ~

Summary The Gardner report, recently published in the UK, showing a correlation between incidence of childhood leukaemia and paternal exposure to ionising radiations (amongst fathers working in nuclear power plants) has added a new element to debates about both the risk factors in nuclear power plants and the relationships between ionising radiations and leukaemogenesis. The epidemiologic and genetic evidence concerning leukaemias is reviewed here and it is concluded that the leukaemogenic agent, whose existence is indicated in the Gardner report, is unlikely to be paternal radiation dose per se but rather exposure to another factor that is correlated with paternal radiation dose received. Introduction The leukaemias and lymphomas are a collection of diseases each of which is essentially the result of an overproduction of one type of haemopoietic cell as a consequence of an abnormality causing the cells in that particular lineage to undergo a failure of differentiation, and hence a continued proliferation. The different leukaemias are very broadly characterised by the type of cell involved, eg a B- or T-lymphocyte, the stage at which maturation is arrested, eg pre-B, and whether the disease progresses rapidly or slowly, ie acute or chronic. Childhood leukaemias, largely acute lymphocytic, or sometimes myelogenous, leukaemias (ALL or AML) and certain closely related lymphomas, are dreadful diseases which, until the advent of modern aggressive therapeutic techniques, were invariably fatal. Now, at least 50 % of sufferers are cured and lead normal lives. The prevalence of childhood leukaemias/ lymphomas in the UK is around 1 in 2000, but the causes of the disease(s) have remained an enigma. Ionising radiations were early shown to be leukaemogenic to adults (where the predominant leukaemia is AML rather than ALL) from studies on individuals exposed to high doses of therapeutic X-rays(') and from the survivors of the atomic bombings at Hiroshima and Nagasaki(2).A number of studies have also revealed an excess of leukaemia amongst the offspring of females exposed to low doses of X-rays during pregnancy("g 3.4) although there have been a few negative reportsCeg including that on the in utero exposed in Hiroshima and

~~

Agent

Population

Authors

Ionising radia tions

Ankylosing spondylitis A-bomb survivors Fetuses

Court Brown and Doll"' UNSCEAR'?) Stewart et a/.(') Monson and MacMaho#)

Organic chemicals Paints/solvents Benzene Gasoline/ Pesticides/ Metal refining

eg aircraft mechanics eg painters eg shoemakers Fetuses

Hicks er a/.'"'

Shu er al.''y'

Virus HTLV-1

Adult T-cell

Yoshida et a/.""'

IARC(~~J' eg Aksoy er u / . ( ? ~ '

Nagasaki(6 but see also 7). 0ther studies (see Table 1)have shown, or claimed, that both adult and childhood leukaemias appear to be associated, to a greater or lesser degree, with occupational exposure to various types of organic chemicals@.') and one specific type of adult human T cell leukaemia is clearly associated with infection with a particular DNA virus, HTLV(''). The fact that cases of childhood leukaemia can occur in clusters was noted long before the widespread advent of nuclear power and in various areas remote from nuclear plants, eg the Vesuvius area in S. Italy(") and Patagonia in Argentina("); such clusters were often thought, but not shown, to be a consequence of viral infection. However, the possibility that some leukaemia clusters might be associated with the ionising radiations emitted from nuclear plants was brought to the forefront in the UK by a Yorkshire television programme broadcast in 1983 which drew attention to an apparent excess of childhood leukaemias in the vicinity of the nuclear reprocessing plant at Sellafield in W. Cumbria. Since that time a number of studies have been undertaken concentrating on 3 areas in the vicinity of nuclear installations (Table 2).

The Nuclear Installations and the Excesses of Childhood Leukaemia/Lymphoma (i) Sellafield, W. Cumbria The Yorkshire TV documentary led directly to the setting up of a government committee which reported in 1984 that there did indeed appear to be an increased frequency of childhood cancers in W. Cumbria('". Among the list of recommendations from the Black committee, all of which have been implemented or set in train, and which included the setting up of the Committee On Medical Aspects of Radiation in the Environment (COMARE) in 1985, were: (i) birth and school cohort studies, the results of which indicated that the leukaemia excess at Sellafield (5 cases observed 2000mSv to raise it 6-8 timed4'), we have no information on viable inherited X-ray-induced mutations in man. However, studies on the mouse show that the minimum X-ray dose to spermatozoa or spermatogonia required to double the spontaneous frequency of a very wide range of different genetic defects is of the order of 150-300 mSv given as an acute exposure(See42) - doses which are at least 15-30 times greater than the doses received in the 6 months prior to conception in the most heavily exposed Sellafield workers. Moreover, in a recent reanalysis of various biological parameters including cancers, in the children born to the survivors of the Japanese atomic bombings, Nee1 and colleagues("344) deduce that the minimal human gametic genetic doubling dose for acute exposures is around 1700-2200mSV, doses vastly in excess of those known to have been received by the parents of the affected Sellafield children. Somatic Genetic Changes in Lymphoid Malignancies Although there is a lack of evidence for a clear heritability of human leukaemias they are nevertheless genetic diseases, but the genetic changes that characterise them are somatic genetic changes. Although detailed pathological findings have not been reported, the majority of the W. Cumbrian childhood leukaemias were acute lymphocytic leukaemias (ALL) - the most common leukaemia of childhood. Approximately 50 YO

of ALLs are characterised by the presence of one of a number of consistent chromosome rearrangements and in a recent review by Mitte1mad4') he lists more than 15 different translocations that appear as specific clonal chromosome aberrations in ALL. Molecular studies of these rearrangements have pin-pointed, at the site of breakage, some of the genes involved in what has been termed the oncogenic conversion of proto-oncogenes normally involved in the control of cellular growth and differentiation. There are two kinds of mechanisms whereby gross chromosomal rearrangements in lymphoid malignancies result in the activation of protooncogenes. These are; first, deregulation of protooncogenes which results in their activation or increased expression and, second, the production of a structural alteration in the proto-oncogene which results in a structural alteration in its gene product and a consequent alteration in its function or activity. (i) Regulation of proto-oncogenes The original, and now classic, example of Burkitt's lymphoma, a lymphoma involving B lymphocytes, where deregulation of expression of the myc oncogene has been shown to occur as a consequence of its juxtaposition to an immunoglobulin (Ig) antigen receptor gene. In the case of Burkitt's lymphoma the myc gene on chromosome 8 may be translocated close to the heavy chain IgH locus on chromosome 14 in the majority (-95 %) of cases, or to the light chain Igrc or Igil loci on chromosomes 2 or 22("' 46). It transpires that human B cell leukaemias and lymphomas frequently have translocations involving chromosome band 14q32.3, the site of the IgH locus. Their T cell counterparts similarly often have an involvement of the T cell locus located at chromosome band 14ql.l and coding for the a chain of the T cell receptor antigen(47). Somewhat less common are rearrangements involving the region coding for the T cell /3 chain receptor on chromosome 7 and, as indicated, in 5 YOof Burkitt's lymphoma cases the Igrc and Igil light chain loci on chromosome 2 and 22 (see Table 5 ) . All the genes at these antigen receptor loci undergo a normal chromosome breakage and rejoining process in the undifferentiated cells in the thymus, to give rise to the mature receptor gene in the lymphocytes. This DNA processing involves a shuffling of segments within each gene complex, an essential natural process that is carried out by a recombinase enzyme utilising recognition sites of 7 o r 9 DNA bases adjacent to each of the V, D or J rearranging segments within the gene complexes. The consistent and specific chromosome abnormalities that correlate with the differing histological and immunological phenotypes of the various leukaemias and lymphomas that involve the fusion of these regions on chromosome 14 or on chromosome 7 to regions on other chromosomes, are clearly the results of errors in this DNA processing mechanism. There is some evidence(48) that the regions of the other chromosomes in the complement that undergo a

Table 5. Oncogenes associated by translocation with an antigen receptor in lymphoid malignancies Oncogene (chromosome/ location)

Protein/location

Neoplasm

myc (8q24)

nuclear 'immediate'

B-lymph

bcl-3 (19q13.1-13.2)

DNA binding 'immediate' (=cdclO) membrane (G protein?)

B-CLL

bcl-2 (18q21)

Lyl-1 (19~13) Ttg (llp15) ZL-3 (Sq31)

DNA binding (Helix-loop-helix) DNA binding (zinc finger) lymphokine

B-lymph T-ALL T-ALL T-ALL B-ALL

Other translocation chromosome 2(Ig4 14q32(IgH) 22(Ig4 14q32 (IgH) 14q32 (W) 7q3s (TC-B) 14qll (TC-(U/4 14q32 (IgH)

Authors see Klein and Klein(46) McKeithan et Ohno et o/.(") CIeary et a1.(h7) Chen-Levy et a1.'5" Haldar et a / . ( h X ) CIeary el u / . ( ~ ' ) Mellentin et a/.""' Rabbitts et a/.'47) McGuire et ai."" Grimaldi and Meeker""

Others uncharacterised with regard to oncogenic potential: TCLl (14q32.1); TCL2 (11~13);TCL3 (9q34.3): TCL3 (1Oq24); TCLS (lp32); bcl-1 (llq13).

translocation (recombination) with the antigen receptor sequences may themselves possess an octameric DNA sequence closely homologous to the chi sequence in bacteria, which is a sequence that promotes recombination. Whatever the mechanism, the consequences in some cases are the juxtaposition of an oncogene next to an actively transcribing receptor sequence and its upregulation and consequent over-expression. A number of rearrangements will not result in the antigen receptor genes being juxtaposed to genes involved with cellular proliferation, and indeed rearrangements involving antigen receptor sequences and other regions of the chromosome complement are seen, but to a far lesser extent, in normal lymphocytes in healthy individuals. (ii) Functional fusion of proto-oncogenes The original, and again a classic, but up until recently the only example of the formation of a fusion gene and chimaeric protein with biochemical properties (tyrosine-kinase activity) which differs from its normal counterpart is the bcr-abl fusion gene, formed as a consequence of a translocation between chromosomes 9 at the locus of the oncogene abl and 22 at the bcr locus, and resulting in the Philadelphia (Ph') chromosome and classic chronic myeloid l e ~ k a e m i a ( ~ ~The , ' ~ ) bcr. abl fusion, resulting in a slightly different fusion gene product with enhanced tyrosine kinase activity, is also observed as a consistent change in a proportion of both AMLs and ALLs("-") and is present in some 5 % of all childhood ALLs("). Earlier this year, 2 reports were providing a second and rather interesting example of a translocation found in 30% of childhood pre-B cell ALLs and involving chromosomes 1 and 19 [t(1:19)(q23:p13.3)]. This translocation results in the production of a fusion protein involving the E2A transcription factor gene on chromosome 19 and a gene, prl, containing a homeobox sequence on chromosome 1. The gene E2A codes for 2 transcription factor

proteins (El2 and E47), that bind to an enhancer sequence within the Igk complex, and is widely expressed. The Prl gene is not normally expressed in pre-B lymphocytes, but the 1:19 translocation results in the expresssion of a fusion chimaeric protein in which the DNA binding domain of E2A is replaced by the DNA binding domain of the homeoprotein. The Prl gene therefore is seen as a candidate p-oncogene that is activated by fusion with the E2A gene. DNA binding proteins acting as transcription factors are coded for by a number of known oncogenes, eg fos,jun, rnyb, and it is evident that the activation of such genes, and of other genes coding for growth factors such as lymphokines (eg IL-3) or cell cycling genes (eg bcl-3), are of cardinal importance in initiating and specifying the phenotypic changes associated with the neoplastic transformations resulting in leukaemias and lymphomas. The mutational changes outlined above are examples of a number of the specific genetic changes associated with the leukaemias. These changes are the consequence of somatic mutations and it is extremely unlikely that many, if any, would be consistent with viability if present as constitutional mutations in an early embryo. One such mutation, the bcr-abl fusion gene producing the p190 fusion protein in ALL, has, however, been successfully introduced as a transgenic microinjected into fertilised mouse eggs. This study(") showed that the bcr-abl construct under the control of the bcr gene promoter was indeed lethal during embryogenesis. However, when the construct was placed under the control of a metallothionein promoter 10 transgenic animals were produced 6 of which rapidly developed aggressive ALL and 2 AML and all died within 10-58 days after birth. The leukaemias produced in the mice were indistinguishable from the human leukaemias associated with the p190 fusion protein. This study underlines the importance of the bcr-abl translocation as a causal factor in human leukaemias, but it does not imply that this single event may be sufficient in itself to result in childhood leukaemias.

Many leukaemias have also associated with them specific additional (eg chromosomes 8, 13, 14 or 21 in ALL) or missing (eg chromosome 7 in AML) chromosomes or chromosome regions and the consistency of these somatic chromosome mutations underlines their importance in the genesis of the neoplastic state(4s). Moreover, the fact that leukaemias can be associated with certain inherited conditions (eg ALL in Ataxia telangiectasia and AML in Fanconi's anaemia) implies that more than one mutation may be necessary before a malignant state is achieved.

What Does the Genetic Evidence Imply in Relation to the Excess Childhood Leukaemias/ Lymphomas in W. Cumbria and Are There Possible Explanations Other Than Induced Mutations in the Sperm of Occupationally Exposed Radiation Workers? The chromosomal mutations referred to above are clearly causal factors in leukaemogenesis, they involve genes concerned with cell growth, development, proliferation and differentiation, but they are confined to the leukaemic cells. As indicated above, these mutational changes would be inconsistent with viability in an early embryo and none have been reported as constitutional changes in the normal cells of leukaemic patients. No cytogenetic information is available on the Sellafield W. Cumbria cases, but in the extremely unlikely possibility of the inheritance of such changes they would be associated with various other abnormalities in addition to leukaemia and none have been reported. Overall therefore, and taking into account the very low external radiation exposure levels to the workers, the absence of a notable increase in inherited abnormalities in the Sellafield children, and the obvious importance of somatic mutations, then the evidence pointing to an inherited mutational change induced in the sperm of workers by low external doses of radiation being the cause of the increased prevalence of leukaemia in their offspring is not supported by our current understanding of the aetiology of leukaemias or of radiation mutagenesis. What other explanations are possible? (i) Chance Unlikely as it may seem, chance cannot be entirely ruled out for the main conclusion of the case control study rests heavily on four of the cases born to Sellafield fathers (see Table 6). As Gardner et al.(16,17)point out, their findings need to be confirmed. Similar case control studies are in progress in relation to the nuclear plant at Dounreay in Scotland and at the Atomic Weapons establishment and ordnance factory at Aldermaston (Berks) and Burghfield (Hants) and the Dounreay results will be published shortly. Additional studies are current in France, Canada and the US, but preliminary negative published data are so far insufficient to draw any conclusions. Perhaps the most important study is that proposed by the National Radiological Protection

Table 6 . Numbers of cases and local controls with relative risks f o r leukaemia and non-Hodgkin's lymphoma in children in relation to external radiation dose (after Gardner et ~ 1 . " ~ ~ " ~ ) Father's accumulated radiation dose

Cases

Controls

RR

95% CL

1-49 mSv 50-99mSv 2100 mSv

4 2 4

41 14 3

0.53 0.95 8.30

0.16-1.78 0.17-5.28 1.36-50.56

Board and the Oxford Childhood Cancer Research Group. The Oxford group have records on over 10000 leukaemic children diagnosed in Britain since 1962 and the NRPB has a register of over 100OOO radiation workers with details of film badge doses. Linking these two registers will be a formidable but necessary task. (ii) Epigenetic changes Can exposure to low dose radiation result in an alteration in gene expression other than by inducing mutation? Considerable evidence is now accumulating for the importance of genomic imprinting in controlling the expression of sets of genes. A differential expression of genes depending upon whether they have been transmitted through the male or female germ line is now well established in both animals and in h ~ m a n s ( ' ~ , Whether ~~). a suppression of the immune response (as a first step prior to a later somatic mutation resulting in leukaemia) could be effected by an imprinting mechanism is not known and there is no evidence that radiation exposure will have an effect on imprinting mechanisms. (iii) Is the registered external exposure on the film badge a surrogate for other more relevant exposures to carcinogens ? The externally recorded film badge dose may be a surrogate for other more relevant exposures to radiation o r indeed to other agents. The suggestion that the most heavily exposed workers inadvertently carry radioactive dusts home to their households is considered most unlikely, but the possibility that these workers may have internal gonadal contamination which could result in sperm being exposed to higher dose levels, or in radioactivity being transmitted to the female partner in seminal fluid, cannot yet be ruled out. Indeed the finding that the risk of prostatic cancer is elevated amongst the most heavily radiation exposed employees at Harwell and Aldermaston has led to the suggestion that some radionuclides may be concentrated in the prostate(59). Radiation workers are, however, not only exposed to radiations. Of the four cases of leukaemia in children born to fathers in the highest radiation dose group one was described as an analytical chemist, one a fitter's mate and the others as process workers. The Sellafield plant consists of a number of buildings and whether these workers all worked in the same building and were exposed to a

common chemical leukaemogenic agent is not known. The evidence for preconceptual exposure to chemical agents in inducing leukaemia in animals, and probably also in humans, is at least as strong as, or indeed stronger than, the evidence for radiation exposure and we have no information on what chemicals are in the environs of the Sellafield workers. A possible carry over of traces of leukaemogenic chemicals, eg decontaminating chemicals, on the bodies and clothing of such workers cannot therefore be discounted and neither can the probability that the leukaemias are induced postnatally and not as a consequence of preconceptional exposure to a mutagen. (iv) Infectious agents Two rather different viruses, the Epstein-Barr (EB) virus in Burkitt’s lymphoma and the human T cell leukaemia virus in adult T cell leukaemia, are associated with specific lymphoid neoplasms in humans; could viruses be involved in the childhood leukaemias? The occurrence of endogenous ecotropic retroviruses is widespread in the genomes of a wide range of mammals, and in many species including chickens, mice, cats, cattle and gibbon apes, it has been shown that leukaemia and lymphomas are a consequence of viral proliferation and transmission(60’61).A number of studies on strains of mice harbouring a provirus with leukaemogenic potential have further shown that activation of latent virus, and the development of leukaemia, may occur following exposure of the To date there is no direct animals to radiations(62363). evidence that retroviruses play a causal role in childhood leukaemia although Kinlen(26927)has provided epidemiological evidence which favours an infectious agent as a significant cause. It may not be irrelevant to note that human lymphoid cells in culture which harbour the E B virus can be made to release large quantities or replicating virus if the cells are exposed to DNA damaging agents, or poor culture conditions, and that viral replication leads to the production of chromosome rearrangements in the host cell. A possible viral aetiology therefore remains an open question.

Conclusion The case-control study by Gardner and his colleagues has revealed an unexpected and, if confirmed, very important possible association between childhood leukaemia and occupational exposure to low levels of external radiations. As they stand, the findings point to the source of the established excesses of childhood leukaemias in the Seascale W. Cumbria region as being related to occupation at the Sellafield nuclear plant. However, the association with low external film badge doses does not of course imply that external radiation dose resulting in germ cell mutations of exposed fathers is the cause of this increase, and indeed the general evidence would strongly suggest that this is not the case. The possibility that the film badge dose is a surrogate

for other exposures, and in particular exposure to chemical agents, appears more likely as indeed is the possibility that the leukaemias are a consequence of fetal or post-natal exposure. Our knowledge of mechanisms in the aetiology of the leukaemias/ lymphomas and of the presence of leukaemic agents other than ionising radiations associated with occupation at the Sellafield plant is insufficient to identify possible causes for the leukaemia excess.

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