Papillary Thyroid Cancer and Polymorphic Variants in TSHR- and RET-Related Genes: a Nested Case-Control Study within a Cohort of U.S. Radiologic Technologists

Thyroid cancer is the most prevalent endocrine malignancy, and papillary thyroid cancer is the most common histologic type. In the United States, ∼90% of all reported thyroid cancer cases are of papillary histology (1). The age-adjusted incidence is low compared with more common cancers but has reportedly increased during the 1990s in several developed countries (1-4). Thyroid cancer occurs more frequently in women, in ∼3:1 ratio, with a median age at diagnosis of ∼46 years (1). The etiology of thyroid cancers remains largely unknown. Some factors, such as height, weight, diet, benign thyroid diseases, reproductive outcomes, and residing in areas of iodine deficiency or excess, have been reported as possible risk factors for thyroid cancer (5-7), but the only established risk factors are a previous history of benign thyroid adenoma and ionizing radiation exposure during childhood (5, 8). Several genetic factors related to thyroid tumorigenesis have been reported (9), but many studies have been limited by small numbers.

The thyrotropin receptor (TSHR) is a key protein in the control of thyroid function and a major thyroid autoantigen. Mutations of the TSHR gene are associated with benign hyperfunctioning thyroid nodules and adenomas (9) and are possibly related to thyroid cancer (10). Rearranged during transfection (RET) gene products are involved in kinase signaling pathways that control the transcription of genes that regulate cell growth and differentiation. The RET gene is a potent oncogene that is associated with several cancers, including thyroid cancer (9, 11-13). We hypothesized that polymorphic variants in TSHR- and RET-related genes might be associated the risk of papillary thyroid cancer. We tested this hypothesis in a nested case-control study within a large cohort of radiologic technologists.

The study population was the U.S. Radiologic Technologists cohort established in the early 1980s to study cancer and other health effects. The details of the U.S. Radiologic Technologists cohort have been reported previously (14). Briefly, the initial cohort, including 146,022 persons (73% women), were radiologic technologists who were certified by the American Registry of Radiologic Technologists for ≥2 years during 1926 to 1982 and resided in the United States. Two postal surveys were administered to this cohort, the first during 1984 to 1989 and the second during 1995 to 1998. The surveys included questions related to work history, health outcomes, and various environmental risk factors. Approximately 110,000 technologists responded to the first and/or second survey.

Thyroid cancer cases (n = 334) were ascertained through self-reports. Living participants reporting thyroid cancer (n = 313) were contacted to obtain consent to acquire medical records to validate the diagnosis. If the participant declined to release medical records (n = 66), or if records verifying the diagnosis could not be obtained (n = 66), the case was excluded from the study. In total, 181 validated papillary thyroid cancer cases were targeted for recruitment, and 167 of them donated blood for analysis. Controls were randomly selected from a pool of controls recruited for ongoing case-control studies of breast, testis, and early-onset cancer within the U.S. Radiologic Technologists cohort. Three controls per case were frequency matched by sex and year of birth (in 5-year strata). In total, 491 control blood samples were included for analysis.

The selection of marker single nucleotide polymorphisms (SNP) was based on review of the published literature and other public databases to cover a part of the TSHR or RET signaling pathways. Genetic variants of interest were EPAC G332S, GFRA1 −193C>G, GFRA3 IVS7+39G>A, RET A45A, RET G691S, RET L769L, RET S686S, TSHR D727E, TSHR IVS1+8651A>G, and TSHR N187N. Samples were genotyped for SNPs using standard Taqman assays, as described previously (15, 16). Primer and probe sequences are available upon request to J.P. Struewing.⁴

All analyses were done using the Intercooled Stata 9.1 statistical software (Stata Co., College Station, TX). SNP frequencies were assessed for deviation from Hardy-Weinberg equilibrium in the controls. Associations between SNPs and risk of papillary thyroid cancer were assessed using unconditional logistic regression analyses. Odds ratios and 95% confidence intervals were calculated as an estimate of the relative risk. We assumed a codominant relationship as numbers permitted; dominant analyses were done when homozygote variant frequencies were <1%. The analyses were adjusted for sex, age, race, smoking habits, exposure to ionizing radiation (including occupational exposure and radiation therapy to the head and neck), year began working as a radiologic technologist, years worked as a radiologic technologist, previous history of benign thyroid conditions, personal and family history of cancer, and age at diagnosis; none of these factors influenced the results (data not shown). Because of the limited effect of the covariates, only the matching variables (age and sex) were included in the final model.

To test any differences between age at diagnosis and gender, we stratified the data by sex and age using two age groups with the cutoff point at the median age of diagnosis.

Distributions for selected variables are shown for 167 papillary cancer cases and 491 controls in Table 1. There were no differences in age at blood collection and race between cases and controls, but the proportion of men among cases (13%) was higher compared with controls (7%) because of a higher nonresponse among male controls. The median age of papillary thyroid cancer diagnosis was 38 years.

Table 2 shows that there were no significant deviations from Hardy-Weinberg equilibrium. The associations between papillary thyroid cancer and polymorphic variants in TSHR- and RET-related genes are shown in Table 2. The results were similar for the univariate analysis and the age- and sex-adjusted analysis. There were no statistically significant associations between papillary thyroid cancer and several polymorphic variants in the overall analysis; however, an association was indicated for RET G691S with a borderline significant trend and potentially also for TSHR N187N, which displayed a borderline significant odds ratio for heterozygous carriers (P_trend = 0.11).

All gender-specific analyses yielded similar results as for the combined analysis (data not shown). For women, the age-stratified odds ratios for RET G691S among those who were under age 38 years at diagnosis were 2.1 (95% confidence interval, 1.2-3.7) for heterozygous carriers and 3.7 (95% confidence interval, 1.1-11.8) for homozygous carriers (P_trend = 0.001). The odds ratios for those who were 38 years or older were 1.2 (95% confidence interval, 0.6-2.3) and 0.8 (95% confidence interval, 0.2-4.2), respectively (P_trend = 0.8). The same age-stratified analysis for men did not indicate any associations between the SNP studied and papillary thyroid cancer risk (data not shown). Stratifying by age at diagnosis did not modify the risks for any other SNPs (data not shown).

The results of this study provide limited support for the hypothesis that polymorphic variants in TSHR- or RET-related genes play a role in the etiology of papillary thyroid cancer. We observed a suggestive decreased risk for papillary thyroid cancer with one SNP in the TSHR pathway (N187N), and we detected a positive association for one SNP in the RET pathway (G691S). The results for RET G691S were especially pronounced among younger women. We have limited support for our findings. Ho et al. (12) reported an association between differentiated thyroid cancer and RET S836S, but not with RET G691S, and Lesueur et al. (17) observed an association between papillary thyroid cancer and RET A45A and RET L769L, findings that we were not able to confirm in this study. A previous study found an association with the RET G691S and medullary thyroid cancer (18). This is, however, a histologic type that shares few features with papillary thyroid cancer.

It is likely that there are different etiologic factors for specific histologic types of thyroid cancer, which may be obscured in a study with multiple histologic types. To increase etiologic homogeneity, we included only confirmed papillary thyroid cancers in our study. This study is, to our knowledge, one of the largest analyzing genotypes in association with papillary thyroid cancer risk but is still limited by a relatively small number of cases; thus, the findings need to be carefully interpreted. The small number of men in the study limits the gender comparison, but it is possible that there is a difference in thyroid cancer etiology between men and women. The fact that thyroid cancer is more common among women may reflect differences in etiology (1).

Selection bias is universally a concern in case-control studies. The exclusion of cases that could not be verified by medical records and participant refusals could potentially have introduced selection bias in our study. The magnitude and direction of this potential bias is difficult to quantify; however, previous analyses that evaluated respondent characteristics in this and other study populations suggest the effect would be small (19). Another concern in genetic analysis is multiple statistical comparisons. Our strategy was to limit the number of SNPs to be tested for associations. The majority of SNPs were chosen based on previous literature indicating an association with several cancers, including thyroid cancer (9, 11-13, 17, 18).

Genetic factors often play an important role in disease development in early life rather than later. Our finding of a stronger association among younger women for RET G691S and thyroid cancer supports this idea. The median age at diagnosis for thyroid cancer in the United States is 46 years (1), which is relatively young compared with many other cancers, and suggests that genetic factors may play an important role with respect to risk.

All control participants in this study were also controls in other nested case-control genetic studies within the cohort and were used for efficiency reasons. The controls were randomly chosen from the pool of controls with blood samples available, and it is unlikely that genotype was related to the chance of being selected. We therefore have no reason to believe that the control selection could affect the estimated variables in any of the studies. Furthermore, most of the allele frequencies among the controls were similar to other populations (12).⁵

In conclusion, our data provide limited support that RET- and TSHR-related genes are associated with papillary thyroid cancer risk. We found a possible association for one SNP in the RET pathway among younger women, but the relatively small sample size suggests caution in interpretation. Thyroid cancer is a rare disease, and it is difficult to obtain biological samples from large numbers of thyroid cancer cases in a defined population, especially for analyses by histologic type. To more definitively assess risks associated with genetic polymorphisms, pooled analyses by combining study populations should be considered.

We thank the radiologic technologists who participated in the U.S. Radiologic Technologists Study, Jerry Reid of the American Registry of Radiologic Technologists for continued support of this study, Diane Kampa and Allison Iwan (University of Minnesota) for data collection and study coordination, Laura Bowen (Information Management Services, Inc., Silver Spring, MD) for data management and computing, and Chris McClure (Research Triangle International, Inc., Research Triangle Park, NC) for tracing and data management.