Microsatellite instability and loss of heterozygosity in radiation-associated thyroid carcinomas of Belarussian children and adults

Hedwig E. Richter1,2,3, Horst D. Lohrer1,2, Ludwig Hieber2, Albrecht M. Kellerer1,2, Edmund Lengfelder1 and Manfred Bauchinger2

1 Radiobiological Institute, University of Munich (LMU), Schillerstraße 42, 80336 Munich, Germany and
2 Institute of Radiobiology, GSF-National Research Center for Environment and Health, Ingolstaedter Landstrasse 1, 85758 Neuherberg, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA from 129 paired thyroid tumorous and non-tumorous tissue samples of Belarussian children (102 patients; age at surgery <=18 years) and adults (27 patients; age at surgery 19–35 years), who had been exposed to radioactive fallout from the Chernobyl reactor accident in 1986, was examined for microsatellite instability (MSI) and loss of heterozygosity (LOH). Twenty-eight microsatellite markers were chosen because of their vicinity to DNA repair genes or genes involved in tumorigenesis as well as regions of chromosomal breakpoints in thyroid tumours. In 40 patients (31% of 129) we detected a total of 73 alterations, 80% of which were classified as LOH and only 20% as MSI. Amongst these 40 patients we identified a subgroup of 11, mainly young female patients (8.5% of 129), exhibiting alterations in at least two microsatellite markers. For comparison we examined samples from spontaneous thyroid carcinomas without radiation history from 20 adult patients from Munich (mean age at surgery 56 ± 13 years). None of the tumour samples investigated showed evidence of alterations in the 28 microsatellite markers tested. Taken together our data indicate an increased instability of microsatellite markers in thyroid cancers from Belarussian patients. At present, it is uncertain whether the increased genome instability observed in Belarussian patients is the result of the exposure to radioactive iodine from the Chernobyl reactor accident or due to the young age of the patients.

Abbreviations: APC, adenomatous polyposis coli; BRCA1/BRCA2, breast cancer genes 1 and 2; DCC, deleted colorectal cancer; FES, feline sarcoma; hMLH1, human MutL-homolog 1; HNPCC, hereditary non-polyposis colorectal cancer; HPRT, hypoxanthine phosphoribosyltransferase; LOH, loss of heterozygosity; MSI, microsatellite instability; MYC, avian myelocytomatosis; RET, receptor-typ tyrosine kinase; RETINT5, receptor-typ tyrosine kinase, intron V; XRCC1 and XRCC5, X-ray repair cross complementing 1 and 5.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the major consequences of the accident at Chernobyl nuclear power plant for public health was an up to 100-fold increased incidence of thyroid cancer in children living in the affected regions (13). The occurrence of these tumours has been linked to the exposure to radioiodine (4), but the mechanisms of radiation carcinogenesis are still poorly understood.

Human cancer has frequently been associated with genetic instability. In the majority of human tumours genome instability was characterized as chromosomal instability (aneuploidy, deletions, amplifications) (5). Only a minority of tumours exhibited mutations on a smaller scale, such as nucleotide instability (6), minisatellite instability (7) or microsatellite instability (MSI) (8), the latter manifested by variations in the copy number of the tandem repeat sequences. Instability of microsatellite sequences was most pronounced in cells from patients with hereditary non-polyposis colon cancer (HNPCC) and is attributed to the loss of mismatch repair genes (9). However, various types of sporadic cancers displayed MSI without evidence of defects in mismatch repair, and thus additional mechanisms must be operative (10).

The analysis of microsatellites also provides information on allelic loss in tumours [loss of heterozygosity (LOH)]. Frequently deleted chromosomal regions suggest the presence of tumour suppressor genes involved in the carcinogenic process. Such allelic loss may occur by mechanisms of hemizygous deletion, chromosomal non-disjunction, mitotic recombination or gene conversion.

Recently Soares et al. (11) investigated 46 benign and malignant thyroid tumours without radiation history at eight loci, mapping to four different chromosomes, and found alterations in three tumours in three and more microsatellite markers. Nikiforov et al. (12), investigating 27 microsatellite markers in 17 samples of tumour and non-tumour DNA of post-Chernobyl pediatric thyroid carcinomas, also found no correlation between MSI and thyroid carcinogenesis. However, minisatellite instability was found in three out of 17 tumours compared with none in 20 sporadic thyroid cancers from patients with no history of radiation exposure. An increased frequency of radiation-associated alterations in minisatellite sequences was described earlier by Dubrova et al. (13) investigating germline mutation of minisatellite loci from children born in highly radiation-contaminated areas after the Chernobyl accident. Compared with a British control group, the mutation rate of Belarussian children was increased up to 2-fold, indicating the impact of radiation exposure on germline mutation rate (13).

Here we present a study of 129 patients who developed thyroid tumours following the exposure to radioactive fallout from the Chernobyl reactor accident. We analysed 28 microsatellite markers situated in the vicinity of DNA repair genes, or genes involved in tumorigenesis, or associated with regions of chromosomal breakpoints, previously determined in post-Chernobyl thyroid cancers (14). We identified 11 tumours of Belarussian children (up to 18 years of age) with alterations in more than two microsatellites, 80% of which were LOHs. In a control group of 20 spontaneous thyroid tumours without radiation history (mean age at surgery 56 ± 13 years) from Munich, we found no alterations in the 28 microsatellite loci investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient groups
MSI and LOH were investigated in paired thyroid tumour and non-tumorous tissue samples from 102 Belarussian children (age at surgery <=18 years) and 27 Belarussian adults (age at surgery 19–35 years). The samples were obtained from the Thyroid Cancer Center of the Institute for Radiation Medicine and Endocrinology, Minsk, Belarus. All patients lived in areas subjected to high radioiodine contamination following the reactor accident in 1986. The mean age at exposure was 3.5 ± 2.5 years for children and 17.3 ± 9.9 years for adults, and the mean ages at surgery were 13.0 ± 2.4 and 27.3 ± 9.2 years, respectively. These data indicate an average latency period of tumour development of 9.5 years for children and 10 years for adults. The female:male ratio for children was 3.1:1 (77/25) and 2.4:1 (19/8) for adults. Histopathological analysis of the 129 tumours revealed 112 papillary carcinomas, two follicular adenomas, five follicular carcinomas, two medullary carcinomas and eight carcinomas without specified histoarchitecture.

For comparison we included in our study a group of 10 female and 10 male thyroid cancer patients (mean age at surgery 56 ± 13 years) from Munich (Martha-Maria Hospital) without radiation history (six papillary carcinomas, six folliculary carcinomas, two medullary carcinomas, two undifferentiated carcinomas, one thyroid carcinoma and three carcinomas without specified histoarchitecture).

Tissue material
Fresh tissue samples were immediately snap frozen in liquid nitrogen and stored at –70°C until use. From most cases, formalin-fixed and paraffin-embedded tissue sections were obtained additionally. Pathological findings according to the TNM classification (15) were provided by the Thyroid Cancer Center, Minsk and the Martha-Maria Hospital, Munich. Paraffin-embedded tissue sections were used for histopathological re-classification.

Isolation of genomic DNA and PCR
Up to 20 mg each of tumour and corresponding non-tumorous tissue was cut, and genomic DNA was extracted using a DNA isolation kit (Qiagen, Hilden, Germany). Extracted DNA samples were employed as templates in PCR experiments in order to amplify microsatellite marker sequence. Oligonucleotide primers corresponding to microsatellite loci were purchased from MWG (Ebersberg, Germany). PCR was performed in 10 µl volumes of a mixture containing 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 100 µM dCTP, dTTP and dGTP, 50 µM dATP, 0,7 µCi [{alpha}-33P]dATP, 0.4 µM each primer, 0.75 U Taq DNA polymerase and 30–50 ng DNA. Thirty-six cycles of 94°C for 1 min, 55–60°C for 1 min and 71°C for 1.5 min were performed, with an initial denaturation step of 94°C for 2 min and a final extension step of 71°C for 7 min using a Perkin-Elmer 9600 GeneAMp PCR System. PCR reaction products were diluted 2:1 with loading buffer [98% formamide, 0.1% xylene cyanol FF, 0.1% bromophenol blue and 10 mM EDTA (pH 8.0)] and denatured for 5 min at 95°C. Subsequently, 4 µl of this solution were electrophoresed through a 6% polyacrylamide gel containing 7 M urea and 32.5% formamide for 2–3 h at 50 W. Following electrophoresis, gels were fixed in 10% acetic acid, washed, dried and exposed to X-ray film for 12–72 h.

MSI and LOH
MSI was defined as a shift of specific allele bands in the autoradiograph. LOH was defined as a total loss or at least 70% reduction of signal density of one allele in the autoradiograph.

The 28 polymorphic markers were located in or near regions of candidate genes (18 markers) or of chromosomal breakpoints (10 markers) previously determined in post-Chernobyl thyroid cancers (14). As shown in Table IGo most microsatellite markers were highly polymorphic dinucleotide (CA)n repeats.


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Table I. Sequences and characteristics of microsatellite oligonucleotide primers
 

    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Instability of microsatellite markers
129 matched DNA samples from non-tumorous and tumorous thyroid tissue were tested with the total of 28 microsatellite markers. Twenty-three microsatellite markers showed alterations in 2–5% of the samples tested; only five markers expressed no instability at all. These five markers were microsatellites associated with the tumour suppressor genes retinoblastoma (RB) and breast cancer gene 2 (BRCA2), the marker BAT26, which has been described as an indicator for the MSI phenotype (16) and two chromosomal markers D13S153 and D2S123. The remaining 23 microsatellite markers showed different degrees of variability, the most unstable of which was human MutL-homolog 1 (hMLH1; seven alterations identified), associated with the human MLH1 mismatch repair gene. However, tumorous DNA samples with alterations of the hMLH1 microsatellite marker showed no increased instability of other markers.

Genomic instability in tumour samples
Instability of one or more microsatellite markers was found in 40 matched pairs (31%) of 129 DNA samples from non-tumorous and tumorous tissues from Belarussian thyroid cancer patients. The group consists of mainly female patients of young age. Of all alterations observed, 80% were classsified as LOHs and were found exclusively in tumour DNA. The remaining 20% were alterations of the copy number of the microsatellite repeat sequences, as deduced from modifications of the migration behaviour of the PCR amplified DNA fragments. Representative autoradiographs are shown in Figure 1Go.



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Fig. 1. Autoradiographs of six different microsatellite loci. APC, FES, HPRT and hMLH1 showed LOH, RET and DCC showed MSI. LOHs were present in tumour but absent in the adjacent non-tumorous tissue. T and N denote DNAs from tumour and non-tumorous tissues respectively.

 
However, there were notable exceptions to the preference of LOHs: all variations of receptor-typ tyrosine kinase (RETINT5), which is associated with the receptor-typ tyrosine kinase (RET) proto-oncogene, were mutations of the marker sequence. Other microsatellite markers with frequent mutations were the ones associated with the X-ray repair cross complementing 1 and 5 (XRCC1 and XRCC5, respectively) DNA repair genes.

Amongst the 40 matched DNA samples we identified a set of 11 samples (8.5% of the total 129) exhibiting alterations in at least two markers (Table IIGo). Only 15 microsatellite markers out of all 23 unstable loci were involved in alterations identified in this group of patients. The loci with the most frequently observed variations were hypoxanthine phosphoribosyltransferase (HPRT; altered in four tumours), feline sarcoma (FES), adenomatous polyposis coli (APC), BRCA2, deleted colorectal cancer (DCC), hMLH1, XRCC5 and D10S1675 (each altered in three tumours). Taking patient data such as age and gender into consideration, we observed that particularly the tumours of young, female patients (<=18 years of age at surgery) showed the highest rate of microsatellite alterations (up to seven altered microsatellites).


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Table II. Microsatellite instability (MSI) and loss of heterozygosity (LOH) for a subset of Belarussian children with at least two unstable microsatellite markers
 
In the reference group of 20 spontaneous thyroid cancers from Munich without radiation history (mean age at surgery 56 ± 13 years), we found no instability of any of the 28 microsatellite loci examined.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 28 microsatellite markers located on 11 chromosomes were investigated in 129 paired tumour and non-tumour DNA samples of thyroid tissue of Belarussian children and adults. The markers included chromosomal regions or genes frequently implicated in human malignancies and involved in mechanisms such as growth control, cell-cycle regulation and DNA repair. Compared with the frequency of microsatellite mutations in HNPCC, our results demonstrate that the phenotype of MSI appeared not to be involved in the development of radiation-associated thyroid tumours nor in spontaneous carcinomas without radiation history. A previous report by Soares et al. (11), investigating eight microsatellite markers in 18 spontaneous thyroid tumours, came to the conclusion that microsatellite instability was no feature of thyroid tumours. A study by Nikiforov et al. (12) of post-Chernobyl pediatric and spontaneous thyroid tumours also concluded that MSI was not involved in thyroid carcinogenesis. However, Nikiforov et al. (12) found somatic cell minisatellite mutations in 18% of pediatric thyroid carcinomas but none in spontaneous thyroid cancers. In support of these findings of increased minisatellite mutations in radiation-induced thyroid cancer, Dubrova et al. (13) determined an increased rate of germline mutations of minisatellites in children born after the accident of Chernobyl into families living in highly contaminated areas. Taken together, both papers suggest the possibility of a radiation-induced cellular system that stimulates minisatellite instability, which in turn contributed to the process of radiation-induced tumorigenesis in the thyroid gland.

In this present study, 40 (31%) thyroid tumours out of 129 showed alterations in at least one microsatellite marker, most of which were LOH. These results correspond to earlier findings of loss of genetic complexity during the development of various other tumours (1720). Within this group of patients we identified a subgroup of 11 (8.5%) patients with two or more altered microsatellite markers. The latter group of patients consists of 10 females and one male with an age at exposure between 1 and 9 years, who developed thyroid carcinoma within 9 years after exposure (age at surgery 13 ± 3 years). This group is somewhat larger than could be expected if microsatellite instability occurred randomly amongst all tumours. It is particularly striking, that within this group there were five patients with four or more alterations. However, each tumour is characterized by a different set of unstable microsatellite markers. Hence, none of the 15 microsatellite markers identified in this subgroup with at least two microsatellite alterations can be used as a `biomarker' for thyroid carcinogenesis.

HPRT, located in the region of the housekeeping gene hypoxanthine phosphoribosyl-transferase, was the most frequently altered microsatellite marker and expressed only LOH. HPRT is one of the three enzymes involved in the synthesis of inositol-monophosphate, a precursor of purine biosynthesis, and has been associated with intestinal and breast carcinomas. It has been suggested that the loss of one allele could result in a perturbation of the dNTP metabolism, leading to early disturbances in DNA transactions (21).

Recent studies have indicated that alterations in microsatellite DNA tend to be associated with proto-oncogenes or tumour suppressor genes, and such alterations at microsatellite loci appeared to play an important role in the development of human cancers (2224). The inactivation of tumour suppressor genes most commonly occurs via mutation of one allele followed by loss of the remaining wild-type allele, or its replacement by a duplicated copy of the mutant allele. In our subgroup of 11 patients with two or more altered microsatellite markers, a high frequency of LOH was observed in oncogenes [FES, avian myelocytomatosis (MYC)] and tumour suppressor genes (APC, BRCA2, DCC). The marker D10S1675, which maps to a region of chromosomal breakpoint 10q26 described earlier (14), also showed a prevalence of LOH. Zedenius et al. (25) and Nikiforov et al. (12) had also detected LOH on 10q (location of markers not defined) in thyroid tumours, indicating the location of a tumour suppressor gene in this region.

In earlier studies, Smida et al. (26) found RET-rearrangements in 65% of papillary thyroid carcinomas of Belarussian children. In our study, we found MSI of RETINT5, which is located in intron 5 of the RET proto-oncogene, in four tumours out of the subgroup of 11 patients with two or more altered microsatellite markers. So far only patient 213 has been included in both studies. Smida et al. (26) detected no RET rearrangement in patient 213; however, an increased level of RET-TK transcript was detected. In this present study, we describe a mutation in the RETINT5 marker sequence in one allele of the tumour sample. Further investigations would be necessary to explore the possibility of a link between the increased transcription of the RET proto-oncogene and the mutation of the microsatellite marker in intron 5.

DNA mismatch repair mechanisms are important factors in the maintenance of genomic stability, and defects in mismatch repair genes were linked to HNPCC development (27). Several genes required for mismatch repair in eukaryotic cells have been identified. We investigated one microsatellite marker, located in the region of hMLH1, which showed LOH in seven tumours (three of the 11 patients subgroup). However, the loss of one allele of hMLH1 was not accompanied by an increased frequency of microsatellite mutations. We concluded that the loss of one allele of hMLH1 did not result in the MSI phenotype and seemed not to be involved in the development of thyroid cancer.

We did not detect any alteration in the 28 microsatellite markers investigated in our reference group of 20 spontaneous tumours. The group involved adult patients (mean age at surgery 56 ± 13 years) without radiation history, with tumours of different types and stages. Considering that 31% of Belarussian cancer patients expressed alterations in at least one microsatellite marker, we expected alterations in six tumours of the reference group. The lack of unstable microsatellites in this reference group was significant (Fisher's exact test; P = 0.0019) and might indicate an induction of genome instability following the exposure to radioactive iodine in the patients from Belarus. Considering the small number of patients without radiation history in comparison with the high number of patients exposed to radiation we can only suggest a link between the exposure to radiation and thyroid carcinogenesis.

Taking together our findings of frequent LOH in tumours from Belarussian patients, but no observed genetic alterations in spontaneous tumours, we conclude that exposure to ionizing radiation initiated genetic instability, which in turn favoured a process of carcinogenesis. Our results also show that young girls in particular were at risk of the induction of thyroid carcinomas following exposure to ionizing radiation. We confirmed previous findings of little importance of MSI in thyroid tumorigenesis in general. However, amongst these patients there is a subgroup of children (mainly girls), whose tumours show signs of genetic instability.


    Acknowledgments
 
The authors thank Nicole Stephan for technical assistance and Herbert Braselmann for help with the statistical analysis of the data. We are most grateful to Dr F.Spelsberg (Martha-Maria Hospital, Munich, Germany), Dr E.Demidchik (Thyroid Center of the Institute for Radiation Medicine and Endocrinology, Minsk, Belarus) and Dr E.Lengfelder [University of Munich (LMU), Munich, Germany] for providing thyroid tissue samples. This study was supported by Bundesamt für Strahlenschutz, Germany, under grant StSch 41 27.


    Notes
 
3 To whom correspondence should be addressed Email: richter{at}gsf.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 9, 1999; revised September 1, 1999; accepted September 9, 1999.