miRNA profiling, detection of BRAF V600E mutation and RET-PTC1 translocation in patients from Novosibirsk oblast (Russia) with different types of thyroid tumors

Nodular thyroid lesions are the most frequent endocrine pathology. Thyroid nodules are diagnosed in over 5 % of the adult population and can be subdivided into benign adenomas or malignant lesions (carcinomas). Carcinomas, requiring mandatory surgery, only make up about 5 % of all neoplasms.

Carcinomas are derived from two types of hormone-producing cells; follicular cells and parafollicular C-cells. More than 95 % of the thyroid carcinomas originate from follicular cells and can be grouped into three main categories: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC). Medullary thyroid carcinomas (MTC) are derived from the parafollicular C-cells, they account for a minority (3 %) of thyroid carcinomas [1, 2].

Three things should be kept in mind: first, even though it was shown that concentration changes of several miRNAs may serve as informative indicators of thyroid nodule malignancy itself, these values are not sufficiently informative for accurate typing of malignant neoplasms (for instance, see [14, 15]); second, lists of miRNAs demonstrating significant expression changes in specific thyroid neoplasms show limited overlap in different reports. This may partially be attributable to both differing efficiencies of detection methods used (as in [16]) and to biases and errors intrinsic to histological and immunohistochemical analysis serving as a reference. Third, a recent study [17] revealed the presence of several discrete subclasses of PTCs, some of these subclasses, statistically less often represented in the total sample, were characterized by expression profiles significantly different from the others.

The purpose of this work is to develop a classifier based on the PCR quantification of a limited number of microRNAs in the surgically excised thyroid tissue. Such an analysis used in addition to histological examination is intended for improving the reliability of typing thyroid neoplasms, including clarification of ambiguous results of histological analysis. Therefore, we planned to pay special attention to controversial cases, where the result of the analysis with the help of the developed classifier was at variance with the result of the histological report.

For this purpose, we planned to assess the difference in the content of several pre-selected miRNAs in the samples from patients with different types of thyroid tumors identified histopathologically; and based on the analysis of the results to develop miRNA classifier for molecular subtyping of thyroid neoplasms.

According to the literature data, increased expression of miR-146b, −221, −222, −181b, and −21 is characteristic of PTC. These, as well as miR-187, were selected as candidates in our work aimed at increasing the accuracy of differential diagnosis of papillary cancer. Fewer data are available for follicular thyroid carcinoma (FTC) and follicular adenoma (FA), and so the major goal here is to discriminate reliably between FA and the rest of the benign nodules. Based on the information presented in Rossing et al. [12], we selected miR-199b-5p and 144-5p so as to enable differential diagnosis of follicular tumors. Next, miR-192 [13] was chosen as a marker that would help discriminate between FTC and FA. Several additional miRNAs were also tested, based on the pronounced histological difference between these tumor types, namely capsular and/or vascular invasion, characteristic of follicular thyroid carcinoma [18]. Follicular subtype is also relatively more prone to metastasis. This property largely results from the invasiveness of cancer cells, which in turn relies on the process of epithelial-mesenchymal transition (EMT). Thus, the above-mentioned additional miRNAs included miR-200a/b, −205, −141-3p and −31, as the former two were reported to control EMT [19, 20] whereas miR-200 is also known to inhibit angiogenesis [21]. miR-141-3p was chosen for it belongs to the same family as miR-200, and miR-31 was demonstrated to negatively regulate cell invasiveness [22]. Finally, consistent with the recent findings, miR-375 was selected as a candidate marker for differential diagnosis of medullary thyroid carcinoma (MTC) [11]. Thus, our final set of miRNAs included miR-221-3p, −222-3p, −146b-5p, −181b-5p, −21-5p, −187-3p, −199b-5p, −144-5p, −192-5p, −200a-3p, −200b-3p, −205-5p, −141-3p, −31-5p, −375.

As additional markers, we intended to use the detection of point mutation in BRAF (V600E) and RET-PTC1 translocation, which most frequently occur in patients with PTC. In the case of contradictory results (a fundamental difference in the histological report and identification of a neoplasm made by the miRNA classifier) detection of one of these markers in the sample may confirm the malignancy of the neoplasm.

Data presented in the Table 7 clearly illustrate possible inaccuracies of histological analysis and a contribution of human bias into the final diagnosis and typing of thyroid malignancies. Although medical expert consultation often makes it possible to correct the error of the initial histological report, our practical experience shows that the human factor remains a significant source of diagnostic inefficiency. Limitations of the “classical” morphological analyses, used in both pre- and post-operative subtyping of thyroid tumors are quite well-recognized. Hence many efforts are currently being undertaken to develop molecular diagnostic tools (including those based on miRNA profiling) to sub-type thyroid neoplasms. The list of proposed molecular cancer-specific markers encompasses point mutations in BRAF and RAS; RET/PTC and PAX8/PPARγ rearrangements; sets of miRNAs, for which significant changes in expression have been established for different types of neoplasms; transcripts of protein-coding genes as well as their products, etc. [33–35].

In our work we did not intend to look for new oncogenic miRNA in thyroid, but used those of them for which the changes in the level have previously been revealed in thyroid neoplasms, selecting a set in such a way as to distinguish between papillary, follicular and medullary thyroid carcinomas and, whenever possible, follicular adenoma.

These differences in the evaluation of the specificity of changes in the content of certain miRNAs with results of other studies are by no means unexpected. Firstly, we used fairly high threshold values, above which changes in the content of miRNAs we considered reliable (for example, for the miR-146b only an increase in comparison with the control more than 8.5 times was seen by us as reliable). To the best of our knowledge, most of the studies in this field were not considered a variation of the procedure of analysis as a whole, from the dissection. We believed that using high threshold values was to increase the specificity of discrimination of tumor types, perhaps at the cost of some desensitization. Secondly, we used for normalization non-tumor adjacent tissue from the same patient, in which the level of certain miRNAs could in itself have been different from the reference values determined for healthy tissue. We chose this strategy because it is more accurate and practically implementable under the surgical removal of the thyroid tissue for the postoperative typing.

On the basis of the obtained data and taking into account the selected threshold values we have managed to develop a classifier based on the analysis of miRNA and enabling the discrimination of neoplasms with sufficiently high specificity. Indeed, we have succeeded in distinguishing different types of carcinomas from each other and from benign neoplasms with sufficient accuracy. Despite the fact that these criteria may have to be updated as new cases are added, they still make it possible to clarify some controversial situations arising at postoperative typing of neoplasms due to the impact of the human factor and because of the ambiguity of the classification itself, especially regarding follicular tumors.

The problems associated with the discrimination of follicular thyroid neoplasms, especially ETC. and FA are all too known. Our current understanding of the data concerning chain of molecular events accompanying the progress of follicular adenoma into carcinoma is far from complete; neither can we tell which morphological characteristics of this transformation can be definitive for a pathologist. For example, the presence of a mutation in a single isoform of the RAS gene can be indicative of an increased potential for the FA transformation to FTC [40, 41], but in follicular adenomas with detected RAS mutations no clear morphological differences have been identified from those, in which such mutations have not been detected. Thus, we speculate that higher resolution of molecular diagnostic tools, as compared to the histological analysis, may underlie the discrepancy between the results of the two approaches.

The fact that molecular classifiers similar to the one proposed by us demonstrate comparatively inferior diagnostic characteristics with regard to follicular neoplasms can be attributed to the heterogeneity of samples classified histologically such as FA, FTC, or FVPTC. On assessing the diagnostic characteristics of our classifier, the histological report served as a reference method, but it is clear that some samples can initially have been mistyped by histological analysis. Also it should be emphasized that in itself the classification of thyroid tumors is by no means established. Discussions are currently underway suggesting its revision due to the new data relating to molecular genetics of thyroid tumors [17, 42].

It should be noted that in most cases of contradictions between the initial histological report and molecular typing, the initial report was a benign tumor and molecular diagnosis confirmed by the secondary diagnosis was cancer (12 out of 20 cases when our classifier indicated cancer). Only in five cases we found somatic mutations indicative of papillary carcinoma and confirming the correctness of typing via miRNA classifier. Undoubtedly, the list of somatic mutations that occur in thyroid cancer is much wider than these two. It should be emphasized, however, that it is generally assumed that only mutations in the BRAF gene and translocation RET-PTC are specific for PTC (and generated by those anaplastic cancers) while other genetic changes often observed in PTC patients (first of all, mutations of RAS) can also be detected in cases of FA and FTC, and, accordingly, their detection would be unable to demonstrate the error of the pathologist's report.

On analyzing the entire sample, we actually detected BRAF (V600E) mutation and translocation RET-PTC1 translocation only in PTC with a frequency corresponding to the literature data: BRAF (V600E) mutation was found in 61.7 % of PTC samples (it was observed in 30–83 % cases, according to [43], RET-PTC1 translocation – in 12 % cases (occurs in 2.5–87 % cases [44–46]). Thus, these two markers together were found in 2/3 cases of PTC. It should be noted that for all samples with detected mutation, the profile of miRNA expression changes, according to our classifier, clearly points to the PTC. In addition, relative levels of several miRNA species in BRAF(V600E)-positive PTCs are significantly higher than in BRAF(V600E)-negative papillary cancers. This trend has been previously noted for several miRNAs from this list (see [47, 48]), however, other studies fail to support these observations [49, 50]. The inconsistencies found thus far may be attributable to both distinct miRNA detection methods, as well as to the peculiarities of the samples.

The proposed miRNA classifier proved to be the most accurate precisely for PTC. It should be emphasized that PTC represents the majority of malignant neoplasms of the thyroid gland. Most errors of pathology reports are associated with its follicular variant. Thus, in our study, in seven cases of initial pathology reports FVPTC was apparently mistaken for FA. We believe that for papillary cancer, the maximum diagnostic reliability both in the preoperative and postoperative typing using the data of the analysis of molecular markers will be able to exclude most of errors arising when only histological report is taken into account.

The high relevance of using molecular markers for improving the reliability of typing thyroid neoplasms is universally recognized nowadays. Thus, since 2015 ATA recommends to identify genetic markers such as mutations of RAS isoforms, mutation of BRAF V600E, translocation RET-PTC, PAX8-PPARγ in refining uncertain cytology results and to take into account the result of the analysis in the further patient management. In relation to clinical relevance of miRNA deregulation the available information is still insufficient, but it should be emphasized that virtually every case of malignant transformation of thyroid tissue is accompanied by changes in the expression profile of miRNAs. At the same time, even for the best studied type of tumors (PTC) it is not always possible to identify the specific somatic mutations, despite the considerable list of mutations known to date [17]. Moreover, the identification of the entire diversity of these genetic changes, including not only point substitutions, but also large-scale restructuring of the genome requires the use of a complex variety of approaches, while the set of diagnostically significant miRNAs is relatively limited, and may be uniformly analyzed by simple methods, such as those based on PCR.

We believe that molecular typing of thyroid neoplasms based on the use of miRNAs offers practical prospects, because it can rely on a limited set of markers while employing such simple method as RT-PCR, i.e., it does not require special equipment and professionals with significant experience. Undoubtedly, additional studies may be required in order to come to a consensus on what set of RNAs is optimal for characterizing of different types of thyroid tumors. However, even with allowance for the obvious limitations of our work due to a limited sampling and not too wide set of the analyzed markers as well as the absence of multicenter validation of the developed classifier, our results show that typing of thyroid neoplasms using microRNA profiling can be a self-sufficient approach. Specific features of such typing enable it to complement morphological typing even without additional analysis of somatic mutations For example, it was found that, our classifier does not distinguish between classical PTC and its follicular variant, (while the set of characteristic somatic mutations for these variants is significantly different) [17], but their distinction by means of histological analysis presents a certain problem, so that the follicular variant of PTC is often confused with follicular neoplasms, including follicular adenomas.

Our data, as well as the results from other groups, argue for the practical feasibility of improved typing of thyroid neoplasms via miRNA profiling by means of simple classifiers in addition to the histological analysis. Even if histological analysis includes the parallel identification of somatic oncogenic mutations, the use of microRNAs may imply additional gain in accuracy.