Iodine-131 dose dependent gene expression in thyroid cancers and corresponding normal tissues following the Chernobyl accident.

The strong and consistent relationship between irradiation at a young age and subsequent thyroid cancer provides an excellent model for studying radiation carcinogenesis in humans. We thus evaluated differential gene expression in thyroid tissue in relation to iodine-131 (I-131) doses received from the Chernobyl accident. Sixty three of 104 papillary thyroid cancers diagnosed between 1998 and 2008 in the Ukrainian-American cohort with individual I-131 thyroid dose estimates had paired RNA specimens from fresh frozen tumor (T) and normal (N) tissue provided by the Chernobyl Tissue Bank and satisfied quality control criteria. We first hybridized 32 randomly allocated RNA specimen pairs (T/N) on 64 whole genome microarrays (Agilent, 4×44 K). Associations of differential gene expression (log(2)(T/N)) with dose were assessed using Kruskall-Wallis and trend tests in linear mixed regression models. While none of the genes withstood correction for the false discovery rate, we selected 75 genes with a priori evidence or P kruskall/P trend <0.0005 for validation by qRT-PCR on the remaining 31 RNA specimen pairs (T/N). The qRT-PCR data were analyzed using linear mixed regression models that included radiation dose as a categorical or ordinal variable. Eleven of 75 qRT-PCR assayed genes (ACVR2A, AJAP1, CA12, CDK12, FAM38A, GALNT7, LMO3, MTA1, SLC19A1, SLC43A3, ZNF493) were confirmed to have a statistically significant differential dose-expression relationship. Our study is among the first to provide direct human data on long term differential gene expression in relation to individual I-131 doses and to identify a set of genes potentially important in radiation carcinogenesis.

RCT Entities:   Population Interventions Outcomes

Introduction

One of the most important health consequences of the 1986 Chernobyl nuclear power plant accident is a dramatic increase in thyroid cancer incidence among those who were children or adolescents at the time [1], [2]. Numerous epidemiological studies have established that this is primarily related to iodine-131 (I-131) exposure from the accident to the thyroid gland [3]–[7]. Despite differences in study populations, designs, and dosimetric approaches, reported estimates of relative risk (RR) per unit of absorbed radiation dose in gray (Gy) for those exposed during childhood are remarkably similar among most studies (3–6 per Gy) and compatible with the risks estimated for thyroid cancer from childhood exposure to external radiation [3]–[8].

The study of radiation carcinogenesis in humans can take advantage of instances where relationships are strong and consistent, as in the situation where the thyroid gland is irradiated at a young age. The opportunity for molecular research of radiation-related thyroid cancer is facilitated by the resources of the Chernobyl Tissue Bank (CTB), which systematically collects biological samples from patients with Chernobyl-related thyroid pathology [9]. Earlier post-Chernobyl studies reported genetic differences between radiation-related and sporadic tumors, including specific RET/PTC gene rearrangements [10]–[12]. However, subsequent analyses utilizing the CTB samples attributed the associations to the younger age of Chernobyl-exposed patients rather than to their radiation exposure [13], [14]. Recent studies using the CTB materials and high throughput technologies have identified new ‘radiation-specific’ genes [15]–[20]. Perhaps the most compelling finding derives from a report comparing exposed and unexposed papillary thyroid cancer (PTC) cases with young age of onset from Ukraine that found a gain of chromosome band 7q11 based on comparative genomic hybridization, confirmed in independent cases by fluorescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qRT-PCR) [20]. However, findings at the gene level are generally inconsistent across studies potentially due to small sample sizes, use of controls from different populations, lack of methodological validation in independent samples, and different analytic approaches. More importantly, none of the previous studies had individual radiation doses assuming that all exposed cases received the same dose. Depending on the true dose-expression relationship, this assumption could cause false positive or false negative associations.

To improve understanding of the molecular consequences of I-131 exposure, we evaluated for the first time differential gene expression in thyroid tissue, defined as a difference in gene expression levels between tumor and corresponding normal thyroid tissue, in relation to individual I-131 thyroid dose estimates. We hypothesized that if dose-related gene expression patterns in tumor tissue truly reflect an important event in radiation carcinogenesis rather than a long-lasting effect of radiation exposure, they should differ from patterns observed in normal tissue; hence our approach of analyzing differential dose-expression relationships in tumor relative to paired normal samples. Our study used RNA specimens from the CTB of patients who underwent thyroid surgery for thyroid cancer in the Ukrainian-American cohort study composed of approximately 13,000 Ukrainian residents <18 years at the time of the accident with individual radioactivity measurements taken within two months after the accident [21].

In the current study, we first conducted an initial screen in half of the cases to identify promising gene candidates that were differentially expressed in tumor and normal tissue specimens in relation to I-131 dose based on whole genome RNA microarrays (phase I). We then validated the top candidates in tumor and normal tissue specimens from the second half of the cases using qRT-PCR (phase II). For the validated candidates we additionally characterized the relationship of gene expression separately in tumor and normal tissue.

Materials and Methods

Patients and Tissue Samples

As a result of four sequential screening examinations, 110 prevalent and incident thyroid carcinomas were diagnosed in the Ukrainian-American cohort between 1998 and 2008 at the Laboratory of Morphology of Endocrine System of the Institute of Endocrinology and Metabolism (IEM, Kiev, Ukraine) [3]. The International Pathology Panel, established in the framework of the CTB project, reviewed all pathological diagnoses. To decrease phenotypic heterogeneity of cases, we excluded 5 follicular and 1 medullary thyroid carcinomas resulting in 104 PTCs eligible for analysis; of these 74 (71%) had stored RNA aliquots from tumor and/or normal tissue in the CTB. Cases included and not included in gene expression analyses were similar in many characteristics including I-131 dose and thus our study sample is likely to be unbiased. All tissue samples were taken intraoperatively after patients signed informed consent forms approved by the institutional review boards (IRB) of the IEM and the Coordinating Center of the CTB project (Imperial College Research Ethics Committee, London, UK). Annual review of the entire project was also provided by the IRB of the National Cancer Institute in the USA.

Detailed operating procedures for the collection, documentation, and processing of frozen tumor and normal thyroid tissue samples are available from the CTB website [22] and were developed jointly with the Laboratory of Morphology of Endocrine System of the IEM and the Wales Cancer Bank.

Dosimetry

Dosimetric methods have been described elsewhere [23]–[25]. Briefly, individual I-131 thyroid doses and their uncertainties were estimated from the combination of thyroid radioactivity measurements, data on dietary and lifestyle habits, and environmental transfer models using a Monte-Carlo procedure with 1,000 realizations per individual [23]. For the analysis, we used the arithmetic mean of each individual’s 1,000 realizations as the best estimate of I-131 dose corrected for thyroid masses typical of the Ukrainian population [3].

RNA Extraction and Quality Control

Full details of RNA extraction can be obtained from the CTB website [22]. In brief, frozen thyroid tissue is homogenised using a tissue lyser (Qiagen, Hilden, Germany). RNA is extracted using Qiagen column-based systems. RNA is frozen at −80°C in standard 5 µg aliquots in 20 µl. Quality and quantity of isolated total RNA is measured spectrophotometrically (NanoDrop, PeqLab Biotechnology, Erlangen, Germany) and RNA integrity is assessed by the 2100 Agilent Bioanalyser (Life Science Group, Penzberg, Germany). For our analysis, we used only RNA specimens with a ratio A260/A280 ≥ 2.0 (Nanodrop) and RNA integrity number (RIN) ≥7.5 or ≥5.5 for whole genome microarray and qRT-PCR analyses, respectively (IMGM Laboratories, Martinsried, Germany).

Although the CTB provided 137 RNA specimens for 71 individuals with PTC, we were able to use 126 paired (tumor/normal) RNA specimens corresponding to 63 individuals (Figure 1). Eleven RNA specimens from eight individuals were excluded due to missing complementary tissue (n = 5) or due to failing our quality criteria specified above (n = 6).

Figure 1

Two stage study design with inclusion and exclusion criteria of cases and selection of gene candidates.

Whole Genome Microarray Analysis (phase I)

Genome wide expression profiling was carried out using the Agilent oligo microarray (4×44 K format) combined with a one-color based hybridization protocol on 64 paired RNA specimens from 32 randomly selected individuals (half the sample set). To assure that individuals selected for microarray gene expression analysis (n = 32) and those remaining for validation analysis by qRT-PCR (n = 31) were similar, we confirmed that their distributions of sex, age, place of residence, and I-131 dose were similar.

Chemicals, kits, and software for the whole genome microarray assay were provided by Agilent Technologies, Waldbronn, Germany. 500 ng of total RNA per specimen (spiked with an internal labeling control) were introduced into an RT-IVT reaction and cDNA was then converted into labeled cRNA by in-vitro transcription step (one color Quick-Amp labeling kit). Quality of labeled non-fragmented cRNA was analyzed (RNA 6000 Nano LabChip kit), and the cRNA was cleaned and quantified (NanoDrop ND-1000 Spetralphotometer). Finally, 1.65 µg of each labeled cRNA sample was fragmented and prepared for one-color based hybridization (gene expression hybridization kit). Hybridization occurred at 65°C over 17 hours on separate microarrays consisting of 41,000 target gene-specific probes (∼20,000 genes) and thousands of control probes. After three washes with increasing stringency, fluorescent signal intensities were detected on the Agilent DNA microarray scanner and extracted from the images using Agilent extraction software. Quantile normalization was applied to the data.

Phase I: Statistical Analysis of Microarray Data and Selection of Gene Candidates for Independent Validation

We analyzed differential gene expression in tumor compared to normal tissue, obtained by subtracting log2 transformed probe signals of the normal tissue (N) from the corresponding tumor tissue (T), log2(T)-log2(N), in relation to I-131 dose estimates. This approach reduces variability in the gene expression. We used the non-parametric Kruskall-Wallis test (P kruskall) to compare differential gene expression across three dose categories (≤0.30, 0.31–1.0, >1.0 Gy) with cut-off points approximately corresponding to the tertiles of dose distribution among cases, and linear regression models with trend test (P linear) for continuous dose. Only those gene transcripts that had a call “present” in at least 50% of RNA specimens from tumor and normal tissue were included in the analysis of differential gene expression (∼15,000). We corrected for multiple comparisons using the false discovery rate (FDR) [26]. As none of the P values remained significant after FDR correction, we adopted the following criteria to select promising candidates for validation and quantification by qRT-PCR: (P kruskall ≤0.001) or (0.001