Epigenetic alterations are potential candidates for the identification of specific markers for cancer detection, diagnosis, and prognosis (1). The utility of epigenetic markers in thyroid cytology should be scoped under the diagnostic, prognostic, and predictive translational extensions, aiming for a more personalized management of patients with a thyroid nodule. For thyroid cytologists, two key questions need to be answered in clinical practice: (1) Is this a case within the group of follicular-patterned lesions? (2) Does this malignant case hold an aggressive morphology? The former question assumes that benign, precursor, or borderline (low-grade malignancies) categories are generally encompassed in a gray zone group of lesions. This is the category that downgrades the diagnostic power of thyroid cytology. To make this first question concrete, if we consider patients with an increased thyroid suppression hormone (TSH) and with a cytology suspicious for follicular or Hürthle cell neoplasm, they should undergo lobectomy or total thyroidectomy, unless a molecular testing would predict a low risk of malignancy. When a surgical decision is made, one needs to consider risk factors for Thyroid carcinoma (TC), clinical and imaging considerations, and patients' informed consent about benefits and risks of diagnostic procedures, including surgery. Surgical complications increase when performing total thyroidectomy compared to lobectomy (2, 3). Prognosis and the need to anticipate risk of recurrence and metastatic disease are clinical challenges that fall under the scope of the second question. Despite the diagnostic power of fine-needle aspiration (FNA) as a gold-standard technique for the presurgical diagnosis of thyroid nodules, the diagnosis of indeterminate categories, especially the lesions falling under atypia of undetermined significance/follicular lesion of undetermined significance and suspicious for follicular neoplasm/follicular neoplasm categories, still remains a challenge in cytopathology practice. Indeed, ~30% of the cases lack the morphological features to provide a definitive classification and are diagnosed as “indeterminate.” With the purpose of reducing unnecessary surgery and predicting malignancy, many panels based on genetic alterations were developed in the past decades (4). These commercially available tests were built on the concept of either rule out or rule in malignancy, based on high-throughput technologies—a targeted next-generation sequencing (NGS) of DNA and RNA. Despite the improvement of diagnosis, ~30% of TC cases are in a genetic “dark zone,” with no well-established driver mutations.

In the past two decades, TC research has started to enlighten the contribution of epigenetic changes to the deregulation of gene transcription and its link with oncogenic pathway activation. The available evidences point to the fact that, beyond genetic factors, the differentiation and proliferation traits of TC cells are strongly influenced by epigenetic alterations. As an example, PTEN promoter presents with hypermethylation in ~50% of papillary thyroid carcinomas (PTCs) and nearly 100% of follicular thyroid carcinomas (FTCs) and follicular adenomas (FAs), suggesting that it may be involved in thyroid tumorigenesis (5). Activating mutations of BRAF in PTCs were linked to altered methylation of other genes, including TIMP3, SLC5A8, DAPK, and RARb2 (6), which were associated with an aggressive behavior in thyroid neoplasms (7). Approximately 30% of benign and malignant thyroid tumors, including anaplastic thyroid carcinoma (ATC), present promoter methylation involving the RAS association family 1A (RASSF1A) tumor-suppressor gene (8–10), known to have a role in regulating several key cell processes, suggesting that this change may occur early in the tumorigenesis.

In this review, our group aimed to provide a brief overview of the epigenetic alterations and their translational potential on thyroid cytology specimens.

The term “epigenetic” was eloquently coined by Waddington (11) in 1942 as describing heritable changes inside the genome leading to an altered gene expression pattern without affecting the main core of DNA. “DNA methylation,” “histone modification,” and “RNA silencing” are considered as main mechanisms of epigenetic regulation of gene expression through the modulation of chromatin structure. In particular, DNA methylation and histone modification are not independent mechanisms; they both act in an orchestrated fashion to regulate chromatin states by using DNA methyltransferases (DNMTs) and a large number of histone-modifying enzymes. Chromatin structure has two different states, “heterochromatin” (a closed chromatin), which is associated with transcriptional repression, and “euchromatin” (an open chromatin) favorable to transcription (12). Each of these effector mechanisms involves enzymes that transfer the modification (“writers”), enzymes that modify or revert a modification (“erasers”), and enzymes that mediate the interactions of proteins or protein complexes with the modification (“readers”), contributing directly or indirectly, through the creation or elimination of protein- or protein complex–binding sites regulating gene expression, to the ectopic transcription of several genes, including oncogenes or proto-oncogenes, or the suppression of tumor suppressor gene transcription. Cancer presents with frequent alterations to the epigenome, including mutations in genes controlling the epigenetic players, silencing several tumor suppressor genes whose roles are implicated in almost all cancer-relevant signaling pathways, such as apoptosis, cell proliferation, cell migration, and DNA repair (13, 14). It has also been well-documented that epigenetic alterations play a significant role in the differentiation and proliferation properties of thyroid cancer (11, 12). Of those, DNA methylation is the most widely studied mechanism, which is responsible for adding a methyl group to the 5′ position of cytosine, predominantly in the context of CpG dinucleotides. Aberrant DNA methylation is the prevalent epigenetic dysregulation in cancer, consisting of both losses (DNA hypomethylation) and gains (DNA hypermethylation) of 5-methyl-cytosine within the CpG dinucleotides. As DNA methylation, histone modification patterns are also dysregulated in cancer. Some histone modifications are associated with gene activation (i.e., trimethylation on histone H3, lysine 4 [H3K4me3]), and others are associated with gene repression (i.e., H3K9me3) (15). Histone modifications are posttranslational modifications, including acetylation, methylation, phosphorylation, ubiquitylation, and glycosylation, among others (15, 16). Histone modifications are the result of the balance between different groups of enzymes, some with antagonist activity. Acetyl groups are added to lysine residues in histone tails by enzymes called histone acetyltransferases causing chromatin decondensation, generally leading to gene activation, and are removed by histone deacetylases (HDACs) (15). Methyl groups are added to lysine and arginine residues by histone methyltransferases, which promote or inhibit transcription, depending on the residue alteration. As an example, methylation of lysine 9 of histone 3 leads to repression, whereas methylation of lysine 4 of the same histone results in gene activation. Methyl groups are removed by histone demethylases (15, 17).

In the last decade, non-coding RNAs (ncRNAs) have been linked to the development and progression of cancer and have been proposed as markers for diagnosis, including of TC (18–24). They can be classified as small interfering RNAs, microRNAs (miRNAs), piwi-associated RNAs, long ncRNAs (lncRNAs), and enhancer RNAs. Of those, the most well-studied ncRNAs are miRNAs, which are known to have either oncogenic or tumor-suppressive roles (25, 26). Interestingly, mutations in DICER1, a member of the RNase III family with a pivotal role in the maturation of miRNAs, have already been described in different human tumors, including TC (27); miRNAs are small single-stranded ncRNAs of ~22 nucleotides transcribed from endogenous DNA and later processed to mature miRNAs, which target and bind to transcripts interfering with protein translation or causing messenger RNA degradation, with an impact on protein production (28).

For DNA methylation detection, cytology smears or cell blocks can be used for bisulfite sequencing, methylation-specific PCR, real-time PCR, immunostaining, and NGS. Advantages and disadvantages of these techniques are outlined in Table 1.

Although all these techniques can be applied on cytology material, the presence of “contaminant cells” can be a practical issue. Studies have shown that 17% of thyroid nodules miss malignant- or benign-specific DNA methylation changes. The absence of epigenetic signatures is frequently linked to lymphocytic thyroiditis. In fact, a group of thyroid nodules with thyroiditis had a DNA methylation pattern very similar to lymph nodes (Figure 1). In addition, 62% of adjacent thyroid tissues and 40% (2 of 5) of malignant tissues with 75% or more of adjacent thyroid tissues were undiagnosable according to Diagnostic DNA Methylation Signature. These data suggest that the presence of cells other than follicular epithelial cells in a specimen may lead to the absence of epigenetic signatures. Such contamination is frequently found in FNA biopsies of thyroid nodules and may include white blood cells, skeletal muscle, blood vessels, or adjacent normal tissue (80).

Despite the improvement of diagnosis through these molecular advances, even the most recent molecular tests present a wide confidence interval for cancer probability, resulting in a lack of established marker(s) that either provide a clear-cut diagnosis or accurately predict the prognosis. Meanwhile, advances in the epigenetic field supporting the differentiation between benign, borderline, and malignant lesions, as well as their role as prognostic factors in malignant neoplasms of various organ systems, such as breast, prostate, lung, or liver, are increasingly acknowledged. Recently, thyroid also started to be studied by various epigenetic techniques. The application of epigenetic-based methods on tissue material of thyroid and various organs has been targeted by researchers in the past two decades, leading to a growing need to establish its applicability to FNA material. Following what has been reported so far, this review amassed the potential usage and adoption of epigenetic-based techniques on thyroid FNA material aimed to better understand the oncobiology of thyroid neoplasia.

SC and AL: conceptualization. SC, AL, and MP: writing – original draft. SC, AL, MP, and VM: writing – review & editing. VM: supervising. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.