Thyroid nodules have been increasingly common in our daily lives and the combination of thyroid ultrasonography and fine needle aspiration biopsy (FNAB) remains the gold standard for identifying or ruling out malignancy with high accuracy (1). However, one out of five aspirates result in the so named “indeterminate samples” or, according to the Bethesda’s Classification System, categories III [atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS)] and IV [follicular neoplasm (FN)/suspicious for follicular neoplasm (SFN)] (2). The rate of malignancy in these indeterminate aspirates varies from 10% to 30% (2). The standard management for these two categories used to be a diagnostic lobectomy around 10 years ago (3). Many groups started to look for less aggressive tools that would reduce the number of indeterminate cytology to minimize the number of resulting unnecessary surgeries, and thus differentiate benign from malignant samples before a surgical procedure was necessary (4).
Nowadays, by mentioning indeterminate nodules, the molecular tests are the ones that stand out. In addition, advances in the understanding of the molecular biology of thyroid tumors, along with advances in genomic technologies have been facilitating its implementation in clinical practice. Due to the fact that such molecular tests have evolved so much and so quickly, currently their applicability serves not only for diagnosis, but also for prognosis and for therapeutic indication purposes (5).
The first molecular markers to gain prominence were point mutations such as BRAFV600E and RAS, and gene fusions like RET/PTC and PAX8/PPARg, in addition to TERT mutations (6). It is known that in most thyroid cancers, mutations are mutually exclusive events, that is, only one of these mutations is found in each tumor (7). When these mutations are used as independent biomarkers, their sensitivity and specificity are too low to be clinically relevant, except for the presence of BRAF and TERT mutations as they are correlated with tumor malignancy (6,8). However, the combination of the analysis of these mutations in a panel has been shown to improve the sensitivity and specificity rates (6,8). Therefore, based on these data, the 7 genes panel was created, and it has inspired groups to create other types of panels that have begun to be commercialized.
Currently, there are 5 commercially available tests being used routinely in clinical practice, particularly in the United States. Most of them, detecting driver mutation or gene fusions (ThyroSeqV3, ThyGeNEXT), or a panel of differential expressed genes (Afirma and Thyroprint) or microRNAs (mirThype, ThyraMIR). Quantitative polymerase chain reaction (qPCR), next generation sequencing (NGS) or RNA-sequencing (RNA-Seq) technologies have been used in these tests to differentiate benign from malignant nodules in indeterminate samples. The prior indication of the molecular tests is for its diagnostic purpose, reaching a sensitivity of 89–94% and specificity of 68–85%, Negative predictive value (NPV) of 94–96% and positive predictive value (PPV) of 47–78% (4) (Table 1). It is important to say that these statistical numbers are influenced by the pretest risk of malignancy (15). Consequently, it is necessary to consider clinical, radiological and cytological information that precedes the molecular test for the indication and correct interpretation of the molecular test results (5). However, the lack of multicenter, prospective, and independent validations of the latest versions of the tests, population-specific validations and, in particular, their high cost, seem to be the biggest barriers towards their total daily applicability routine in many countries (4).
|Characteristics||ThyroSeq (9)||ThyGeNEXT (10)||Afirma (11)||mirTHYpe (12)||ThyroPrint (13)||Epigenetic imprinting biomarkers (14)|
|Molecular material analyzed||Mutations, fusions, CNA||Mutations, fusions, miRNA||Gene expression||miRNA||Gene expression||Imprinting genes|
NPV, negative predictive value; PPV, positive predictive value; CNA, copy number alteration; NGS, next generation sequencing; qPCR, quantitative polymerase chain reaction; RNA-Seq, RNA-sequencing.
Little has been seen or discussed in the latest International Congresses about an improvement in the diagnostic point of view of these molecular tests. It seems that we have been satisfied with the diagnostic accuracy of the known driver mutations or gene rearrangements, of the few differentiated expressed genes or panel of miRNAs, by reaching sensitivities of 89–95% and specificities of 68–85%. Therefore, what we have been seeing lately is an effort to understand the prognostic role of the known molecular markers, and how they can help us to guide a targeted therapy, rather than trying to improve their diagnostic accuracy (5).
Nevertheless, the article published by Xu et al. (14) with the title: “The high diagnostic accuracy of epigenetic imprinting biomarkers in thyroid nodules”, published on the Journal of Clinical Oncology on November 15, 2022 took us out of our comfort zone regarding what we had known about molecular panels for indeterminate thyroid nodules at what we had considered “sufficient” as diagnostic sensitivity, specificity, PPV and NPV. The group surprised us with their data about a panel of epigenetic imprinting biomarkers in indeterminate thyroid nodules reaching a sensitivity of 100%, a specificity of 91.5% (95% CI: 86.4–96.5%), a PPV of 96.5% (95% CI: 94.4–98.6%), and a NPV of 100% in the prospective validation, with a diagnostic accuracy of 97.5% (384/394; 95% CI: 95.9–99.0%). That means: a complete novel category of molecular marker and different technology that had never been used before when compared to the current validated molecular tests. In addition, reaching numbers that had not been reached by any commercially available molecular test before (Table 1).
It is defined that epigenetics are changes in gene expression which can be inherited, and that are not related to variation in the DNA sequence (16). Historically, the word “epigenetics”, a term introduced by Conrad Waddington in 1942, had been used to describe events that could not be explained by genetics (DNA), and defined epigenetics as “the branch of biology that studies the causal interactions between genes and their products, which give rise to the phenotype” (16). Over the years, numerous biological phenomena, some considered unusual and unexplored, have been grouped under the category of epigenetics. Currently, epigenetics is defined as the biological processes that promote gene expression variation, and which cannot be changed in the nucleotide sequence of the gene (17).
Translating the genetic information of “epigenetic imprinting biomarkers’’ detected by QCIGISH technology into practical words: In normal somatic cells, maternal and paternal alleles of an imprinted gene are differentially methylated, thus one of allele is silenced and the other, activated. However, in the case of cancers, both alleles are expressed due to the activation of the imprinted gene. This is called “loss of imprinting” (18). So, the method discussed in the paper by Xu et al. (14) analyses the non-coding intronic RNA aiming to visualize the transcription loci of the imprinted gene. For that, the QCIGISH method uses different colours to characterize the different structures of the nuclei: blue, red and brown. The different allelic expressions of the imprinted genes are quantified based on the transcription signals. Normal cells will show 1 or no colour, however, aberrant expressions will show more than one color signal (19). Putting together, the principle of the QCIGISH methodology is to visualize, quantify and confirm pathologic allele expression of the investigated imprinted genes (14).
Using this method, Xu et al. (14) demonstrated the diagnostic value of the expression status of three imprinted genes candidate to differentiate benign from malignant indeterminate thyroid samples: guanine nucleotide-binding protein, alpha-stimulating complex locus (GNAS), growth factor receptor-bound protein (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN). The prospective multicenter study, involving 394 patients, showed an overall diagnostic accuracy for combined cases of Bethesda III, IV and V of 98.2%.
Quite impressive numbers, especially when compared with other methodologies like the well-known and established PCR, NGS, RNA-Seq or type of genetic material studied (DNA, RNA, miRNA). However, in front of a novelty, other questions start to emerge: (I) Is QCIGISH technique accessible and also established as PCR or NGS? (II) Is it an easy reproducible method, easily applicable to other countries? (III) Is it cost-effective? (IV) Do we need high-qualified specialists to carry out the analyses? (V) How about the pre-analytical phase: how complex is the preparation of the material for this technology? (VI) Is it easy to add this test to a clinical routine?
Perhaps a novel category may be arriving, or at least, new horizons are opening up in this era of precision medicine, and what was thought to be well established may change in the next few years. And while we thought that an “all stages” test: diagnostic, prognostic and therapeutic was enough, the epigenetic may be presenting us another tool, perhaps much more powerful than the previous ones, mainly to the diagnosis of indeterminate samples. We will anxiously await the answers to the above questions and further studies to help us define the applicability of this new molecular marker.
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