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Solid cancers after therapeutic radiation – can we predict which patients are most at risk?
Clinical Oncology (2004) 16: 429e434 doi:10.1016/j.clon.2004.04.008
Overview Solid Cancers After Therapeutic Radiation e Can We Predict Which Patients Are Most at Risk? G. A. Thomas South West Wales Cancer Institute, Singleton Hospital, Swansea, Wales, UK Key words: Post-Chernobyl thyroid cancer, Hodgkin’s disease, single nucleotide polymorphism Received: 13 April 2004 Accepted: 19 April 2004
Introduction
The improvement in the treatment of cancer means that patients are surviving for longer. The consequence of this increased survival is that late effects of radio- and chemotherapy can now be observed. Many therapeutic options for cancer combine radiotherapy with chemotherapy. Both result in damage to the DNA, which, in most affected cells within the cancer, may lead to cell death. However, if exposure of normal cells to these mutagenic and clastogenic agents leads to damage insufficient to result in cell death, but which is inappropriately repaired, the first step on the pathway to development of a second malignancy may be taken. Cancer results from the successive acquisition of defects in growth regulatory mechanisms within a clone of cells. Subsequent propagation of a clone of cells harbouring a particular mutation increases the likelihood of a second mutation occurring in a sub-clone of cells containing the first mutation. Therefore, propagation of clones bearing a mutated phenotype is a key step in the eventual development of a clinically significant neoplasm. For this reason, it might be expected that exposure to mutagenic stimuli early in life, in tissues that are still undergoing developmental growth, would increase the risk of developing of neoplasia later in life.
Effect of Age at Exposure on Development of Neoplasia after Radiation Exposure
A number of studies have investigated the effect of age at exposure to radiation on the subsequent risk of developing of thyroid neoplasia. Ron et al. [1] published a pooled analysis of seven studies in which radiotherapy had been given to the head and neck for a variety of reasons. These included enlarged thymus or tonsils, tinea capitis, and a variety of childhood cancers. The results clearly showed that radiation below the age of 5 years was associated with Author for correspondence: G. A. Thomas, South West Wales Cancer Institute, Singleton Hospital, Swansea SA2 8QA, Wales, UK Tel: C44-1792285-407; Fax: C44-1792-285-201; E-mail: [email protected] 0936-6555/04/000000C06 $35.00/0
a significantly elevated risk of developing thyroid cancer. The most dramatic association between age at exposure and radiation has been observed in those areas of Belarus, Ukraine and Russia that were exposed to radioiodine in fallout from the accident at the Chernobyl Nuclear Power Plant in April 1986 [2,3]. Here, there has been a large increase in thyroid cancer in people aged 0e5 years at the time of the accident. Thyroid cancer is usually rare in children (of the order of 0.5e1.5 per million per year [4]). However, people aged less than 1 year at the time of the accident and who were residents in the most highly contaminated areas of Belarus showed a relative risk of 237, whereas people aged 10 years showed a relative risk of only 6 d a 40-fold difference in sensitivity [5]. The Chernobyl accident produced the largest peacetime release of beta-emitting isotopes of radioiodine into the atmosphere [6]. The exposure to the population was largely through inhalation and the ingestion of contaminated milk. The thyroid is the only organ in the body to concentrate and bind iodine. Although the whole body radiation dose was relatively low, the concentration of radioiodine by the gland (individual thyroid doses are estimated to be between 0.02 and 3 Gy in Ukraine and Belarus), coupled with the sensitivity of the maturing thyroid gland of small children, have probably resulted in the particular sensitivity of this young cohort to the health consequences of the accident. We do not yet know the long-term effects of the exposure of such a large cohort of children (about 10 million) to low-dose radiation. Although the thyroid seems to be a particularly radiosensitive organ, brain tumours and haematological malignancies have also been shown to develop with longer latencies [7] after larger therapeutic doses of radiation to the head and neck region in childhood. A recent study of long-term survivors of Ewing’s sarcoma found three second malignancies in a total of 42 patients irradiated at a mean of 16.8 years (average follow-up 25 years). Two of these (a thyroid cancer and a breast cancer) were not considered to be due to radiation therapy, as the cancer sites were not located within the radiation field. However, the third d a spindle cell sarcoma d was concluded to be of a radiogenic cause [8].
Ó 2004 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
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Secondary Primary Cancers after Treatment for Hodgkin’s Disease
One of the most common types of malignancy that involves the use of chemotherapy, radiotherapy, or both, in children, but which also has a high cure rate is Hodgkin’s lymphoma. Among the secondary primary tumours observed in Hodgkin’s patients are melanoma, sarcoma, lung, thyroid and breast cancer. The mantle field used for radiation therapy in Hodgkin’s disease (HD) inevitably leads to exposure of the breast, lungs and thyroid to radiation. As with all radiation-induced tumours, post-HD second primary malignancies have a long latency, and those who were young at the time of treatment for HD show a greater risk. For girls with HD, the greatest risk seems to be the development of breast cancer [9e13]. In a report of a British cohort of 5519 patients with HD treated during a 30-year period (1963e1993), younger age at radiotherapy for HD (under 25 years) was shown to be an important risk factor for all second primary malignancies. The standardised incidence ratio for female breast cancer falls from 14.4 (95% CI 5.7e29.3) in women aged under 25 years at treatment to 1.6 (95% CI 0.5e3.7) in women aged 25e44 years at treatment [12]. The youngest cohort (1380 children diagnosed between 1955 and 1986) from the American Late Effects Study Group, aged below 17 years at treatment for HD, reported 42 cases of breast cancer in 30 patients (29 women, 1 man) after a median follow-up of 17 years [11]. Twelve patients presented with bilateral breast cancer or subsequently developed bilateral disease. The median age at diagnosis of HD was 14.2 years (range 6.6e15.6 years), and age at diagnosis of breast cancer was 32 years (range 16.3e42.7 years). The median time to development of breast cancer was 18.1 years (range 4.3e28.3 years). Interestingly, this cohort reiterates the findings of earlier studies, which have shown that cohorts aged 0e5 years at diagnosis of HD are at higher risk of developing thyroid cancer [11]. The results of these studies suggest that there is a window of time during which particular tissues may be most sensitive to the carcinogenic effects of radiation exposure. For thyroid, this is early childhood, when the follicular cells of the gland are probably at its most active stages of division. In the breast, development of the ducts and lobules takes place around puberty, between the ages of 10 and 16 years, and this is when this tissue seems to be at its most radiosensitive. Effect of Dose on Development of Breast Cancer After Radiation
A clear doseeresponse relationship exists between to the development of breast cancer after exposure to the atomic bomb in Japan and exposure to therapeutic radiation. Breast cancer incidence among survivors of the atomic bomb in Hiroshima (where doses were higher) show a 25% greater risk of developing breast cancer than those in Nagasaki [14]. This doseeresponse relationship seems to be linear [15], and no low-dose threshold can be defined below
which there is no risk [16]. Patients with HD who were treated with a combination of radio- and chemotherapy and received 38.5 Gy or more showed a relative risk of 4.5 times compared with patients receiving less than 4 Gy. For those treated with radiation only, breast cancer risk increased with increasing dose of radiation given [13]. Although the dose given to HD patients is lower now than in the past, it is unlikely that there is a completely safe and effective dose that can be given.
Other Factors Influencing Risk of Breast Cancer after Hodgkin’s Disease
With breast cancer, factors other than age at exposure clearly play a role in the overall risk of developing of a second neoplasm as a result of exposure to radiation. Studies of the survivors of the atomic bomb indicate that a younger age at first pregnancy seems to decrease breast cancer risk [15]. Van Leeuwen et al. [13] recently reported a caseecontrol study of women treated for HD, including 48 women who developed breast cancer and 175 matched controls that did not. This important study also provided evidence that chemotherapy that resulted in induced premature menopause (MOPP: mechlorethamine, vincristine, procarbazine and prednisone) may protect against the development of breast cancer after radiotherapy, suggesting that exposure to oestrogen acts as a ‘promoting’ factor for the radiation-induced damage. The use of radiotherapy is diminishing, and HD is now usually treated by a combined radiotherapy and chemotherapy approach. The use of newer chemotherapy regimens that spare ovarian function may in fact lead to an increase in secondary breast cancer relative to that seen in patients treated with radiotherapy and chemotherapy that destroys ovarian function.
Secondary Primary Malignancies after Radiotherapy for Other Cancers
Although the risk of second malignancy after radiotherapy is comparatively lower when patients receive their treatment in adulthood, a number of studies suggest that a significant risk may exist. A recent study [17] reported an elevated risk of developing bone and soft tissue, colorectal, salivary gland cancer and leukaemia after 131-I therapy for thyroid cancer, demonstrating a doseeresponse relationship. In addition, Deutsch et al. [18] reported an increased incidence of lung cancer after radiotherapy for breast cancer within a randomised clinical trial setting, confirming an earlier study by Zablotska and Neugut [19]. Both suggest that it is the volume of lung tissue exposed to radiation that is a critical factor in determining risk, and that there is a long post-radiation latency for developing of these tumours. The move to the use of intensity-modulated radiation therapy (IMRT) may also lead to an increase in the development of second primary malignancies. With IMRT, rather than conventional radiotherapy, a larger
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volume of normal tissue is exposed to lower doses. A significant increase in the mutational load to normal tissues surrounding the cancer may occur, leading to an increase in the risk of developing a second cancer within the radiation field. It has been estimated that IMRT may double the incidence of second malignancies compared with conventional radiotherapy from about 1%e1.75% for patients surviving 10 years [20].
Is There a Genetic Predisposition to the Development of Secondary Cancer after Radiation Therapy?
There is great interest in understanding whether patients who develop breast cancer after Hodgkin’s lymphoma have a genetic predisposition to developing breast cancer after radiation exposure or whether breast cancers that develop in these patients show a radiation signature. Radiation exposure leads to the formation of double-strand breaks in DNA. Two pathways are used in eukaryotic cells to repair this type of damage: the homologous recombination pathway and the non-homologous end-joining pathway (Fig. 1). There is a common initiation for the pathway, when a protein complex made up of NBS1, MRE11 and RAD50 binds to the DNA at the site of the double-strand break. The pathways then separate using a number of other protein complexes to bring about repair. BRCA1 and BRCA2, germline mutations shown to be implicated in breast cancer risk are involved in the homologous repair pathway. Extensive research into the identification of mutations in other members of this pathway has taken place; knocking out the function of particular proteins might predispose to a risk of developing breast and other cancers. It now seems increasingly probable that polygenic effects may occur and that single nucleotide polymorphisms (SNPs), which on their own do not significantly affect protein function, may, in combination, give rise to sufficiently dysregulated DNA repair to lead to predisposition to cancer. A recent report suggests that variants in other genes (XRCC2, XRCC3 and LIG4) concerned with DNA double-strand break repair may play a role in the susceptibility of breast cancer in general [21]. In the small studies that have so far been reported, no correlation has been observed with mutations of the ATM gene sequence [22] in patients with breast cancer after treatment for Hodgkin’s lymphoma. However, large studies would be required to determine whether SNPs in a single or multiple gene lead to predisposition to the development of a second primary neoplasm after radiation therapy. The recent recall of patients with Hodgkin’s disease treated under the age of 35 years with supradiaphragmatic radiotherapy presents an opportunity to collect samples of DNA from these patients to be used in the search for markers that may predict which patients are at risk from the development of subsequent cancers. It is most probable, if a pattern of genetic predisposition can be discerned in these patients, that this will relate to the chance of developing a second neoplasia, but other factors such as lifestyle (e.g. smoking for lung cancer), and the environmental and
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hormonal factors already associated with breast cancer risk, may also play an important role in the risk of site-specific secondary cancer. However, there is a possibility that SNPs in genes involved in oestrogen synthesis [23], in combination with SNPs in DNA repair, may significantly elevate the risk of developing breast cancer. Although the search for germline predisposition has not yet yielded good candidate genes for general breast cancer risk, searching for such associations in clearly defined patient groups, such as those treated with radiotherapy for HD, may yet pay dividends. Study of this cohort may reveal genes that predispose to radiation sensitivity, although the possible involvement of genes in the non-homologous end-joining pathway in this is controversial [24e26]. Collection of material for biological sub-studies planned for the SUPREMO clinical trial will greatly help our scientific understanding of genes that predispose to radiosensitivity. Combining information from this clinical trial with studies on patients with second primary malignancies after HD may delineate a common set of ‘radiosensitivity’ genes.
Is There a Molecular Signature in Cancers Associated with Radiation Exposure?
There has been speculation that cancers that can be ascribed to a radiation cause may show a ‘radiation signature’. So far, only a few small studies of molecular changes in tumours after treatment for HD have been reported. In one study, no loss of heterozygosity for BRCA1 or BRCA2 was found in breast cancers [27]. Another study was unable to identify frame-shift mutations in microsatellite sequences at the coding regions of a number of candidate genes in either breast or lung cancers [28]. This study did, however, find an increased overall frequency of microsatellite alterations, suggesting widespread genomic instability. A similar observation was made in a series of paediatric thyroid cancers after Chernobyl [29]. The study of the molecular biology of post-Chernobyl thyroid cancer has been greatly facilitated by the establishment of a pathologically reviewed tissue bank, the Chernobyl Tissue Bank (http://www. chernobyltissuebank.com). Most post-Chernobyl thyroid cancers are papillary carcinomas. Papillary carcinomas are known to be associated with rearrangements of the ret oncogene [30], although the overall frequency of different ret rearrangements varies considerably among studies. Early studies on post-Chernobyl thyroid cancer reported that there was a higher than expected frequency of ret rearrangement, particularly ret PTC3, in paediatric papillary carcinomas, suggesting that some ret rearrangements might be regarded as a marker for radiation exposure [31e33]. However, few statistically valid studies have been published of ret rearrangement in non-Chernobyl-associated paediatric thyroid cancers [34,35], making substantiation of the association of ret rearrangements with radiation exposure difficult. It is important to remember that the correlation between molecular biology and pathology is not absolute: in all of the series published so far, a substantial proportion
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Fig. 1 e Genes involved in breast cancer risk and DNA double-strand break and repair pathways. Genes that have been associated with increased risk of breast cancer in non-radiation-exposed cohorts are outlined in red. It is possible that polymorphisms in these genes may play a greater role in the risk of developing breast cancer after exposure to radiation.
(30e50%) of the papillary cancers do not harbour a ret rearrangement. A variety of different techniques has been used to assess the frequency of ret rearrangement and, although this may explain the variation in frequency of ret rearrangement among studies, there still remains a large proportion of papillary carcinomas for which alternative molecular pathways need to be identified. Moreover, a few studies have shown ret rearrangements in benign tumours associated with radiation exposure [36,37], however, other studies have failed to substantiate these findings [38],
adding further uncertainty to the specific association of ret rearrangement with papillary thyroid cancer after exposure to radiation. Despite evidence that ret is able to transform the follicular cell in vitro, evidence from transgenic mice suggests that other oncogenic mutations must be required for tumour development [39,40]. The clinical relevance of ret rearrangement in post-Chernobyl papillary carcinoma still remains unclear. Some studies in adults have suggested that the presence of ret rearrangement may confer a better
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prognosis, but other studies have found the opposite [41e43]. In addition, it has also been argued that ret rearrangements are not found in all cells in post-Chernobyl papillary carcinomas, and that cells harbouring the rearrangement may be clustered. This suggests either a polyclonal origin for these tumours, or that ret rearrangement is a later event in thyroid papillary carcinogenesis than had previously been thought (Unger et al., personal communication). In addition, the B-raf oncogene has recently emerged as the most commonly mutated oncogene in papillary carcinoma in adults. The frequency varies in a number of studies from 36e69% in adult papillary thyroid carcinoma [44e46], including one study on Ukrainian tumours (Thomas et al., personal communication). The frequency of B-raf mutation in post-Chernobyl cases (aged under 18 years at operation) is much lower (of the order of 7%) and does not seem to be significantly different from that observed in sporadic childhood thyroid papillary carcinoma [47, Lima et al., personal communication]. This finding is perhaps not surprising as B-raf and ret oncogenic alterations seem to be mutually exclusive in the series published thus far. However, it is clear that all cases that are negative for B-raf in young-onset papillary cancer are not necessarily positive for ret rearrangement, and that there are as yet unidentified oncogenic changes in these tumours. Post-Chernobyl papillary thyroid carcinomas, in common with non-radiation-associated childhood papillary carcinomas do not harbour ras mutations, [48] p53 mutations [48,49] or show microsatellite instability [48]. Most studies focus on assessing frequency of one or two oncogenes in a series of tumours. Studies using cDNA array enable the examination of multiple changes in gene expression. One recent cDNA array study suggests that the overall profile of post-Chernobyl papillary cancers are similar to papillary carcinomas from Belgium and France (Detours et al., personal communication), and that, even using expression analysis of a large number of genes, no radiation signature has yet been identified. To further complicate matters, there is now evidence that the morphology of post-Chernobyl papillary cancer is changing with time after the accident [50,51]. Further studies will need to be carried out to ascertain whether the change in morphology is accompanied by a change in the molecular profile of post-Chernobyl thyroid cancer, but early indications are that this is so. It is important to separate the effects of an aetiological agent on the molecular biology of a tumour from the effects of the microenvironment in which the tumour develops. Cancer develops as a series of changes in the growth control of a cell that confers a clonal growth advantage. It is perhaps not surprising that the molecular changes that provide a growth advantage for tumour cells in the child may be different from those required to provide a growth advantage in the adult, given the huge changes in the hormonal environment between infancy and adulthood. In the breast, there is evidence that oestrogen receptor negative tumours are more common in younger (premenopausal) women than older (post-menopausal) women
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[52]. The experience with studying thyroid cancer after Chernobyl shows that it will be necessary to ensure that studies on the molecular biology of second primary malignancies after treatment for HD include appropriate age-matched controls, before conclusions can be drawn either regarding radiation signatures within tumours, or inherited mutations that lead to a predisposition to development of breast cancer after radiation. Conclusion
In conclusion, it is unlikely that ‘radiation signatures’ in second malignancies after radiation therapy will be identified. In effect, the tissue microenvironment in which the tumour develops exerts pressure to select those mutations that confer clonal growth advantage. It is, therefore, more probable that the molecular biology of tumours will harbour signatures that owe more to the age of the patient at clinical presentation than to the aetiological agent. It is more probable that germline SNPs in DNA repair genes may lead to an overall susceptibility to develop second cancers after radiation. Other ‘environmental’ effects, such as oestrogen exposure for breast cancer, which may be modulated by chemotherapy effects on the ovary, may moderate the propensity to develop cancers at a given tissue site after exposure to iatrogenic radiation. Development of highquality collections of biological material from patients who develop second primary malignancies after radiation would greatly increase the chance of being able to assess accurately an individual’s response to radiotherapy for their primary malignancy and the risk of subsequent development of a second radiation-associated primary malignancy. Acknowledgements. The European Commission; National Cancer Institute of the USA, the Sasakawa Memorial Health Foundation of Japan; the World Health Organization for their sponsorship of the Chernobyl Tissue Bank; Professor R.C.F. Leonard for academic and personal support.
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Overview Solid Cancers After Therapeutic Radiation e Can We Predict Which Patients Are Most at Risk? G. A. Thomas South West Wales Cancer Institute, Singleton Hospital, Swansea, Wales, UK Key words: Post-Chernobyl thyroid cancer, Hodgkin’s disease, single nucleotide polymorphism Received: 13 April 2004 Accepted: 19 April 2004
Introduction
The improvement in the treatment of cancer means that patients are surviving for longer. The consequence of this increased survival is that late effects of radio- and chemotherapy can now be observed. Many therapeutic options for cancer combine radiotherapy with chemotherapy. Both result in damage to the DNA, which, in most affected cells within the cancer, may lead to cell death. However, if exposure of normal cells to these mutagenic and clastogenic agents leads to damage insufficient to result in cell death, but which is inappropriately repaired, the first step on the pathway to development of a second malignancy may be taken. Cancer results from the successive acquisition of defects in growth regulatory mechanisms within a clone of cells. Subsequent propagation of a clone of cells harbouring a particular mutation increases the likelihood of a second mutation occurring in a sub-clone of cells containing the first mutation. Therefore, propagation of clones bearing a mutated phenotype is a key step in the eventual development of a clinically significant neoplasm. For this reason, it might be expected that exposure to mutagenic stimuli early in life, in tissues that are still undergoing developmental growth, would increase the risk of developing of neoplasia later in life.
Effect of Age at Exposure on Development of Neoplasia after Radiation Exposure
A number of studies have investigated the effect of age at exposure to radiation on the subsequent risk of developing of thyroid neoplasia. Ron et al. [1] published a pooled analysis of seven studies in which radiotherapy had been given to the head and neck for a variety of reasons. These included enlarged thymus or tonsils, tinea capitis, and a variety of childhood cancers. The results clearly showed that radiation below the age of 5 years was associated with Author for correspondence: G. A. Thomas, South West Wales Cancer Institute, Singleton Hospital, Swansea SA2 8QA, Wales, UK Tel: C44-1792285-407; Fax: C44-1792-285-201; E-mail: [email protected] 0936-6555/04/000000C06 $35.00/0
a significantly elevated risk of developing thyroid cancer. The most dramatic association between age at exposure and radiation has been observed in those areas of Belarus, Ukraine and Russia that were exposed to radioiodine in fallout from the accident at the Chernobyl Nuclear Power Plant in April 1986 [2,3]. Here, there has been a large increase in thyroid cancer in people aged 0e5 years at the time of the accident. Thyroid cancer is usually rare in children (of the order of 0.5e1.5 per million per year [4]). However, people aged less than 1 year at the time of the accident and who were residents in the most highly contaminated areas of Belarus showed a relative risk of 237, whereas people aged 10 years showed a relative risk of only 6 d a 40-fold difference in sensitivity [5]. The Chernobyl accident produced the largest peacetime release of beta-emitting isotopes of radioiodine into the atmosphere [6]. The exposure to the population was largely through inhalation and the ingestion of contaminated milk. The thyroid is the only organ in the body to concentrate and bind iodine. Although the whole body radiation dose was relatively low, the concentration of radioiodine by the gland (individual thyroid doses are estimated to be between 0.02 and 3 Gy in Ukraine and Belarus), coupled with the sensitivity of the maturing thyroid gland of small children, have probably resulted in the particular sensitivity of this young cohort to the health consequences of the accident. We do not yet know the long-term effects of the exposure of such a large cohort of children (about 10 million) to low-dose radiation. Although the thyroid seems to be a particularly radiosensitive organ, brain tumours and haematological malignancies have also been shown to develop with longer latencies [7] after larger therapeutic doses of radiation to the head and neck region in childhood. A recent study of long-term survivors of Ewing’s sarcoma found three second malignancies in a total of 42 patients irradiated at a mean of 16.8 years (average follow-up 25 years). Two of these (a thyroid cancer and a breast cancer) were not considered to be due to radiation therapy, as the cancer sites were not located within the radiation field. However, the third d a spindle cell sarcoma d was concluded to be of a radiogenic cause [8].
Ó 2004 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.
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Secondary Primary Cancers after Treatment for Hodgkin’s Disease
One of the most common types of malignancy that involves the use of chemotherapy, radiotherapy, or both, in children, but which also has a high cure rate is Hodgkin’s lymphoma. Among the secondary primary tumours observed in Hodgkin’s patients are melanoma, sarcoma, lung, thyroid and breast cancer. The mantle field used for radiation therapy in Hodgkin’s disease (HD) inevitably leads to exposure of the breast, lungs and thyroid to radiation. As with all radiation-induced tumours, post-HD second primary malignancies have a long latency, and those who were young at the time of treatment for HD show a greater risk. For girls with HD, the greatest risk seems to be the development of breast cancer [9e13]. In a report of a British cohort of 5519 patients with HD treated during a 30-year period (1963e1993), younger age at radiotherapy for HD (under 25 years) was shown to be an important risk factor for all second primary malignancies. The standardised incidence ratio for female breast cancer falls from 14.4 (95% CI 5.7e29.3) in women aged under 25 years at treatment to 1.6 (95% CI 0.5e3.7) in women aged 25e44 years at treatment [12]. The youngest cohort (1380 children diagnosed between 1955 and 1986) from the American Late Effects Study Group, aged below 17 years at treatment for HD, reported 42 cases of breast cancer in 30 patients (29 women, 1 man) after a median follow-up of 17 years [11]. Twelve patients presented with bilateral breast cancer or subsequently developed bilateral disease. The median age at diagnosis of HD was 14.2 years (range 6.6e15.6 years), and age at diagnosis of breast cancer was 32 years (range 16.3e42.7 years). The median time to development of breast cancer was 18.1 years (range 4.3e28.3 years). Interestingly, this cohort reiterates the findings of earlier studies, which have shown that cohorts aged 0e5 years at diagnosis of HD are at higher risk of developing thyroid cancer [11]. The results of these studies suggest that there is a window of time during which particular tissues may be most sensitive to the carcinogenic effects of radiation exposure. For thyroid, this is early childhood, when the follicular cells of the gland are probably at its most active stages of division. In the breast, development of the ducts and lobules takes place around puberty, between the ages of 10 and 16 years, and this is when this tissue seems to be at its most radiosensitive. Effect of Dose on Development of Breast Cancer After Radiation
A clear doseeresponse relationship exists between to the development of breast cancer after exposure to the atomic bomb in Japan and exposure to therapeutic radiation. Breast cancer incidence among survivors of the atomic bomb in Hiroshima (where doses were higher) show a 25% greater risk of developing breast cancer than those in Nagasaki [14]. This doseeresponse relationship seems to be linear [15], and no low-dose threshold can be defined below
which there is no risk [16]. Patients with HD who were treated with a combination of radio- and chemotherapy and received 38.5 Gy or more showed a relative risk of 4.5 times compared with patients receiving less than 4 Gy. For those treated with radiation only, breast cancer risk increased with increasing dose of radiation given [13]. Although the dose given to HD patients is lower now than in the past, it is unlikely that there is a completely safe and effective dose that can be given.
Other Factors Influencing Risk of Breast Cancer after Hodgkin’s Disease
With breast cancer, factors other than age at exposure clearly play a role in the overall risk of developing of a second neoplasm as a result of exposure to radiation. Studies of the survivors of the atomic bomb indicate that a younger age at first pregnancy seems to decrease breast cancer risk [15]. Van Leeuwen et al. [13] recently reported a caseecontrol study of women treated for HD, including 48 women who developed breast cancer and 175 matched controls that did not. This important study also provided evidence that chemotherapy that resulted in induced premature menopause (MOPP: mechlorethamine, vincristine, procarbazine and prednisone) may protect against the development of breast cancer after radiotherapy, suggesting that exposure to oestrogen acts as a ‘promoting’ factor for the radiation-induced damage. The use of radiotherapy is diminishing, and HD is now usually treated by a combined radiotherapy and chemotherapy approach. The use of newer chemotherapy regimens that spare ovarian function may in fact lead to an increase in secondary breast cancer relative to that seen in patients treated with radiotherapy and chemotherapy that destroys ovarian function.
Secondary Primary Malignancies after Radiotherapy for Other Cancers
Although the risk of second malignancy after radiotherapy is comparatively lower when patients receive their treatment in adulthood, a number of studies suggest that a significant risk may exist. A recent study [17] reported an elevated risk of developing bone and soft tissue, colorectal, salivary gland cancer and leukaemia after 131-I therapy for thyroid cancer, demonstrating a doseeresponse relationship. In addition, Deutsch et al. [18] reported an increased incidence of lung cancer after radiotherapy for breast cancer within a randomised clinical trial setting, confirming an earlier study by Zablotska and Neugut [19]. Both suggest that it is the volume of lung tissue exposed to radiation that is a critical factor in determining risk, and that there is a long post-radiation latency for developing of these tumours. The move to the use of intensity-modulated radiation therapy (IMRT) may also lead to an increase in the development of second primary malignancies. With IMRT, rather than conventional radiotherapy, a larger
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volume of normal tissue is exposed to lower doses. A significant increase in the mutational load to normal tissues surrounding the cancer may occur, leading to an increase in the risk of developing a second cancer within the radiation field. It has been estimated that IMRT may double the incidence of second malignancies compared with conventional radiotherapy from about 1%e1.75% for patients surviving 10 years [20].
Is There a Genetic Predisposition to the Development of Secondary Cancer after Radiation Therapy?
There is great interest in understanding whether patients who develop breast cancer after Hodgkin’s lymphoma have a genetic predisposition to developing breast cancer after radiation exposure or whether breast cancers that develop in these patients show a radiation signature. Radiation exposure leads to the formation of double-strand breaks in DNA. Two pathways are used in eukaryotic cells to repair this type of damage: the homologous recombination pathway and the non-homologous end-joining pathway (Fig. 1). There is a common initiation for the pathway, when a protein complex made up of NBS1, MRE11 and RAD50 binds to the DNA at the site of the double-strand break. The pathways then separate using a number of other protein complexes to bring about repair. BRCA1 and BRCA2, germline mutations shown to be implicated in breast cancer risk are involved in the homologous repair pathway. Extensive research into the identification of mutations in other members of this pathway has taken place; knocking out the function of particular proteins might predispose to a risk of developing breast and other cancers. It now seems increasingly probable that polygenic effects may occur and that single nucleotide polymorphisms (SNPs), which on their own do not significantly affect protein function, may, in combination, give rise to sufficiently dysregulated DNA repair to lead to predisposition to cancer. A recent report suggests that variants in other genes (XRCC2, XRCC3 and LIG4) concerned with DNA double-strand break repair may play a role in the susceptibility of breast cancer in general [21]. In the small studies that have so far been reported, no correlation has been observed with mutations of the ATM gene sequence [22] in patients with breast cancer after treatment for Hodgkin’s lymphoma. However, large studies would be required to determine whether SNPs in a single or multiple gene lead to predisposition to the development of a second primary neoplasm after radiation therapy. The recent recall of patients with Hodgkin’s disease treated under the age of 35 years with supradiaphragmatic radiotherapy presents an opportunity to collect samples of DNA from these patients to be used in the search for markers that may predict which patients are at risk from the development of subsequent cancers. It is most probable, if a pattern of genetic predisposition can be discerned in these patients, that this will relate to the chance of developing a second neoplasia, but other factors such as lifestyle (e.g. smoking for lung cancer), and the environmental and
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hormonal factors already associated with breast cancer risk, may also play an important role in the risk of site-specific secondary cancer. However, there is a possibility that SNPs in genes involved in oestrogen synthesis [23], in combination with SNPs in DNA repair, may significantly elevate the risk of developing breast cancer. Although the search for germline predisposition has not yet yielded good candidate genes for general breast cancer risk, searching for such associations in clearly defined patient groups, such as those treated with radiotherapy for HD, may yet pay dividends. Study of this cohort may reveal genes that predispose to radiation sensitivity, although the possible involvement of genes in the non-homologous end-joining pathway in this is controversial [24e26]. Collection of material for biological sub-studies planned for the SUPREMO clinical trial will greatly help our scientific understanding of genes that predispose to radiosensitivity. Combining information from this clinical trial with studies on patients with second primary malignancies after HD may delineate a common set of ‘radiosensitivity’ genes.
Is There a Molecular Signature in Cancers Associated with Radiation Exposure?
There has been speculation that cancers that can be ascribed to a radiation cause may show a ‘radiation signature’. So far, only a few small studies of molecular changes in tumours after treatment for HD have been reported. In one study, no loss of heterozygosity for BRCA1 or BRCA2 was found in breast cancers [27]. Another study was unable to identify frame-shift mutations in microsatellite sequences at the coding regions of a number of candidate genes in either breast or lung cancers [28]. This study did, however, find an increased overall frequency of microsatellite alterations, suggesting widespread genomic instability. A similar observation was made in a series of paediatric thyroid cancers after Chernobyl [29]. The study of the molecular biology of post-Chernobyl thyroid cancer has been greatly facilitated by the establishment of a pathologically reviewed tissue bank, the Chernobyl Tissue Bank (http://www. chernobyltissuebank.com). Most post-Chernobyl thyroid cancers are papillary carcinomas. Papillary carcinomas are known to be associated with rearrangements of the ret oncogene [30], although the overall frequency of different ret rearrangements varies considerably among studies. Early studies on post-Chernobyl thyroid cancer reported that there was a higher than expected frequency of ret rearrangement, particularly ret PTC3, in paediatric papillary carcinomas, suggesting that some ret rearrangements might be regarded as a marker for radiation exposure [31e33]. However, few statistically valid studies have been published of ret rearrangement in non-Chernobyl-associated paediatric thyroid cancers [34,35], making substantiation of the association of ret rearrangements with radiation exposure difficult. It is important to remember that the correlation between molecular biology and pathology is not absolute: in all of the series published so far, a substantial proportion
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Fig. 1 e Genes involved in breast cancer risk and DNA double-strand break and repair pathways. Genes that have been associated with increased risk of breast cancer in non-radiation-exposed cohorts are outlined in red. It is possible that polymorphisms in these genes may play a greater role in the risk of developing breast cancer after exposure to radiation.
(30e50%) of the papillary cancers do not harbour a ret rearrangement. A variety of different techniques has been used to assess the frequency of ret rearrangement and, although this may explain the variation in frequency of ret rearrangement among studies, there still remains a large proportion of papillary carcinomas for which alternative molecular pathways need to be identified. Moreover, a few studies have shown ret rearrangements in benign tumours associated with radiation exposure [36,37], however, other studies have failed to substantiate these findings [38],
adding further uncertainty to the specific association of ret rearrangement with papillary thyroid cancer after exposure to radiation. Despite evidence that ret is able to transform the follicular cell in vitro, evidence from transgenic mice suggests that other oncogenic mutations must be required for tumour development [39,40]. The clinical relevance of ret rearrangement in post-Chernobyl papillary carcinoma still remains unclear. Some studies in adults have suggested that the presence of ret rearrangement may confer a better
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prognosis, but other studies have found the opposite [41e43]. In addition, it has also been argued that ret rearrangements are not found in all cells in post-Chernobyl papillary carcinomas, and that cells harbouring the rearrangement may be clustered. This suggests either a polyclonal origin for these tumours, or that ret rearrangement is a later event in thyroid papillary carcinogenesis than had previously been thought (Unger et al., personal communication). In addition, the B-raf oncogene has recently emerged as the most commonly mutated oncogene in papillary carcinoma in adults. The frequency varies in a number of studies from 36e69% in adult papillary thyroid carcinoma [44e46], including one study on Ukrainian tumours (Thomas et al., personal communication). The frequency of B-raf mutation in post-Chernobyl cases (aged under 18 years at operation) is much lower (of the order of 7%) and does not seem to be significantly different from that observed in sporadic childhood thyroid papillary carcinoma [47, Lima et al., personal communication]. This finding is perhaps not surprising as B-raf and ret oncogenic alterations seem to be mutually exclusive in the series published thus far. However, it is clear that all cases that are negative for B-raf in young-onset papillary cancer are not necessarily positive for ret rearrangement, and that there are as yet unidentified oncogenic changes in these tumours. Post-Chernobyl papillary thyroid carcinomas, in common with non-radiation-associated childhood papillary carcinomas do not harbour ras mutations, [48] p53 mutations [48,49] or show microsatellite instability [48]. Most studies focus on assessing frequency of one or two oncogenes in a series of tumours. Studies using cDNA array enable the examination of multiple changes in gene expression. One recent cDNA array study suggests that the overall profile of post-Chernobyl papillary cancers are similar to papillary carcinomas from Belgium and France (Detours et al., personal communication), and that, even using expression analysis of a large number of genes, no radiation signature has yet been identified. To further complicate matters, there is now evidence that the morphology of post-Chernobyl papillary cancer is changing with time after the accident [50,51]. Further studies will need to be carried out to ascertain whether the change in morphology is accompanied by a change in the molecular profile of post-Chernobyl thyroid cancer, but early indications are that this is so. It is important to separate the effects of an aetiological agent on the molecular biology of a tumour from the effects of the microenvironment in which the tumour develops. Cancer develops as a series of changes in the growth control of a cell that confers a clonal growth advantage. It is perhaps not surprising that the molecular changes that provide a growth advantage for tumour cells in the child may be different from those required to provide a growth advantage in the adult, given the huge changes in the hormonal environment between infancy and adulthood. In the breast, there is evidence that oestrogen receptor negative tumours are more common in younger (premenopausal) women than older (post-menopausal) women
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[52]. The experience with studying thyroid cancer after Chernobyl shows that it will be necessary to ensure that studies on the molecular biology of second primary malignancies after treatment for HD include appropriate age-matched controls, before conclusions can be drawn either regarding radiation signatures within tumours, or inherited mutations that lead to a predisposition to development of breast cancer after radiation. Conclusion
In conclusion, it is unlikely that ‘radiation signatures’ in second malignancies after radiation therapy will be identified. In effect, the tissue microenvironment in which the tumour develops exerts pressure to select those mutations that confer clonal growth advantage. It is, therefore, more probable that the molecular biology of tumours will harbour signatures that owe more to the age of the patient at clinical presentation than to the aetiological agent. It is more probable that germline SNPs in DNA repair genes may lead to an overall susceptibility to develop second cancers after radiation. Other ‘environmental’ effects, such as oestrogen exposure for breast cancer, which may be modulated by chemotherapy effects on the ovary, may moderate the propensity to develop cancers at a given tissue site after exposure to iatrogenic radiation. Development of highquality collections of biological material from patients who develop second primary malignancies after radiation would greatly increase the chance of being able to assess accurately an individual’s response to radiotherapy for their primary malignancy and the risk of subsequent development of a second radiation-associated primary malignancy. Acknowledgements. The European Commission; National Cancer Institute of the USA, the Sasakawa Memorial Health Foundation of Japan; the World Health Organization for their sponsorship of the Chernobyl Tissue Bank; Professor R.C.F. Leonard for academic and personal support.
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