Association of CYP2D6*4 gene polymorphism with early papillary thyroid carcinoma

Introduction

Although thyroid carcinomas constitute only 1% of all malignancies in humans; they are responsible for more than 90% of all endocrine cancers [1]. The majority of thyroid cancers are epithelial tumors that arise from thyroid follicular cells. They are mainly classified into three subtypes: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC). Differently, medullary thyroid carcinoma (MTC) derives from thyroid parafollicular (C) cells [2].

PTC, the commonest type of endocrine malignancy, is a differentiated cancer and its incidence has risen a lot in recent decades [3]. PTC is classified into three major variants. Those are classic (CVPTC), follicular (FVPTC) and tall cell variants (TCVPTC). The genetic alterations of the signaling proteins in the mitogen-activated protein kinase (MAPK) pathway, including RET, ALK, RAS and BRAF are closely related with PTC [4]. Many additional tests used together with thyroid specimens have focused on biomarkers in this signaling pathway [5]. Although more advanced molecular tests are convenient, BRAF mutation is the most crucial biomarker for the diagnosis of PTC [6]. The molecular mechanisms that participate in PTC tumorigenesis and progression are not fully understood. Thus, elucidation of the underlying molecular mechanisms will be useful for the advancement of new diagnosis and treatment strategies for PTC.

Cytochrome P450s (CYPs) are mostly located in the liver and metabolize xenobiotics to non-toxic or carcinogenic metabolites [7]. CYP2D6 (debrisoquine hydroxylase) is a member of CYP family and the CYP2D6 gene takes place on chromosome 22q13.1 [8]. CYP2D6, takes part in the metabolism of drugs and bioactivation of several procarcinogens and neurotoxins [9]. The gene encoding the CYP2D6 enzyme is very polymorphic with more than 100 variants, and the allele frequencies vary between ethnic populations [10]. People may be sorted as poor, intermediate, extensive, or ultrarapid metabolizers depending on the CYP2D6 genotype combinations [11]. Among the variant alleles with non-functional activity, CYP2D6*4 (G1934A) is the most common one [10], [12]. A single nucleotide polymorphism at the splice site of intron 3 and exon 4 leads to the development of a deficient enzyme variant (rs3892097; c.1846G>A) (also called as CYP2D6*4 isoform) and consequently result in a poor metabolizer status [13]. The poor metabolizer genotype has been shown to have effect on susceptibility to various types of cancers including head and neck [14], acute lymphoblastic leukemia (ALL) [15], prostate [16] and breast cancer [17]; but only a few studies have been studied for thyroid cancer [18]. In the present study, we aimed to evaluate the contribution of CYP2D6*4 polymorphism to the susceptibility of PTC in a Turkish population considering their demographic and histopathological characteristics.

Materials and methods

Results

All of the patients with PTC had occult tumors. The patients had no secondary tumors. The mean age was 42.37 ± 10.52 years in patients with PTC and 40.33 ± 8.32 years in controls (p>0.05). The female to male ratio was greater in the PTC group when compared with the controls but there was no significance (in PTC group: 71/26, in control group: 79/41; p>0.05). Our patient group consisted of two variant subgroups: CVPTC and FVPTC. The distributions of tumor variants for gender and age were alike. The frequency distributions of PTC patients according to tumor stages were 31 (32%) PT1, 15 (15.4%) PT2, 39 (40.2%) PT3 and 12 (12.4%) PT4. The ‘A’ allele (CYP2D6*4) frequency was higher in patients who had encapsulated tumors, but it was not statistically significant (56.2 vs. 33.3%, p=0.111). The frequency distribution of histopathologic parameters among papillary thyroid carcinoma cases is given in Table 1. The differences were not significant between the PTC patient and control groups, when age and sex distributions were analyzed (p>0.05). The distributions of genotypes and alleles of CYP2D6 are shown in Table 2 and Figure 1(a). The frequency of ‘A’ allele was greater in cases with PTC than in control group (OR: 1.995, 95% CI: 1.060–3.752, p=0.031). Moreover the ‘A’ allele frequency was higher in patients with age ≤50 years when compared to those with age >50 (OR: 2.380, 95% CI: 1.191–4.755, p=0.013). The age-dependent distributions of genotypes and alleles of CYP2D6 are shown in Figure 1(b).

Figure 1:

Age-independent (a) and age-dependent (b) risk stratification of CYP2D6*4 polymorphism in patients with papillary thyroid carcinoma (PTC).

The distribution of CYP2D6 polymorphism was not consistent with HWE (p<0.05) in both patients and control groups. The ‘A’ allele frequency was higher among the patients who had lymphocyte infiltration in their tumor-surrounding tissues, but there was no statistical significance (p=0.211). Genotype and allele frequencies were not significantly different between patients with the classical and the follicular variant of PTC. CYP2D6 genotype and allele frequencies were not significantly different between patients with the CVPTC and FVPTC or between patients at early (I/II) and late (III/IV) stage tumors. BRAF V600E mutation analysis of the same patient group was analyzed in our previous study [19]. Fifty-four (55.67%) of the PTC patients had BRAF V600E mutation. According to the histological type of the tumor, the BRAF V600E mutation was present in 65.67% of cases of CVPTC (44 of 67) and in 33.33% (10 of 30) of FVPTC. The frequency of BRAF V600E mutation was statistically higher in CVPTC compared to FVPTC (X ²=8.44, OR: 3.739, 95% CI: 1.501–9.312, p=0.004).

The relationship between BRAF V600E and CYP2D6 genotypes is given in Table 3. No association was found between BRAF V600E mutation and CYP2D6*4 alleles.

Discussion

PTC incidence rate has increased worldwide, in recent decades. The development of new diagnostic techniques provided detection of even small tumors [23]. Humans are exposed to many different chemicals as a result of modern life. The biotransformation of those xenobiotics are mainly done through two enzymatic pathways: phase 1 and phase 2. A plenty of polycyclic aromatic hydrocarbons, pesticides, herbicides, and drugs are metabolized by CYPs [24]. CYPs are highly polymorphic and there is an inter-individual variability in the capacity to detoxify carcinogens or activate procarcinogens.

CYP2D6*4 which is the most prevalent polymorphism among Caucasians and creates a deficient protein [25]. Poor metabolizers are not capable of producing functional CYP2D6 enzymes. Therefore the substrates of the enzyme accumulate to a large extent without being metabolized [26]. Insufficient capacity to detoxify toxic compounds causes DNA and cellular damage due to the genomic instability resulting from the construction of chemical elements linked to DNA and protein molecules. Consequently, the possibility of developing cancer increases [27].

First, in 1984, Ayesh et al. reported that CYP2D6 enzyme activity had an effect on cancer, in lung cancer patients [28]. Thereafter, many studies were conducted on investigating the influence of CYP2D6 on susceptibility to various kinds of cancer. However, the influence of CYP2D6 allelic variants in different types of cancer has remained controversial. While some studies suggested a possible role for the poor metabolizer genotype in the development of cancer, other studies could not find evidence for this. The CYP2D6*4 allele was not reported to effect the susceptibility to bladder [29] or gastric cancer [30] risk in Tunisian and Chinese patients, respectively. No association was reported with CYP2D6*4 and the risk of acute myeloid leukemia AML and ALL in Turkish cases [31], but a risk was found in Brazilian ALL cases [15]. The influence of CYP2D6*4 on breast cancer susceptibility is also controversial [17], [32]. On the other hand, CYP2D6*4 allele frequency was significantly reduced in patients who had tumors in their pituitary glands [25].

The CYP2D6*4 allele was found to have a protective effect on susceptibility to PTC in Portuguese [18]. In contrast, we found that presence of CYP2D6*4 polymorphism might be a risk determinant for PTC in patients with especially age ≤50 years in Turkish population. The diversity in ethnic and geographical conditions as well as the genetic backgrounds and environmental exposures may have an impact on the risk of PTC associated with the CYP2D6 genotype. Lemos et al. reported that, although they have different morphological structures, the two major variants CVPTC and FVPTC had similar CYP2D6 genotype and allele frequencies Likewise, no difference was found between the early and late tumor stage, or multifocality [18]. The findings of the current study were similar to their findings.

In this study, the distribution of CYP2D6*4 allele frequency was not concordant with Hardy–Weinberg equilibrium (HWE) in both patients and control groups. This may be due to the relatively small size of our study groups [33]. Moreover, according to the Hardy–Weinberg principle, factors such as mutations, migration, lack of natural selection and non-random mating may cause genotypic and allele frequencies in the population to remain constant from generation to generation [34].

Silveira et al. reported that, since the CYP2D6 gene takes a role in the detoxification of carcinogenic compounds; and thus due to the lack of enzymatic activity genotoxic metabolites gets accumulated in phase I metabolism process thereupon the risk of ALL increases [15]. Here, we agree with Silveira et al. that presumably the accumulated genotoxic metabolites forming as a consequence of poor metabolizing enzyme activity may take a role in the development of PTC.

Conclusion

To our knowledge, there is no study other than ours investigating the relationship between CYP2D6*4 allele and histopathological characteristics and risk of PTC, in Turkish patients. Our results may provide new insight to understand the molecular causes of thyroid cancer susceptibility. The major limitation of this study was the relatively low number of participants in the patient and control groups. Despite the fact that the results may provide clinical impact, our data needs confirmation by increasing the number of cases in the study.