Background
Radioactive iodine (RAI) therapy is a very effective treatment, significantly increasing the life expectancy of differentiated thyroid cancer (DTC) patients [
1]. However, about 60% of metastatic tumors (corresponding to less than 5% of all thyroid cancers) will become radioiodine-refractory (RAI-R), with a major impact on patient prognosis. In particular, the 10 years survival rate significantly decreases in patients without any
131I uptake at diagnosis compared to patients with initial
131I uptake but persistent diseases, and even more if compared to RAI-avid (RAI-A) metastatic thyroid tumors [
2].
According to American Thyroid Association (ATA) Guidelines [
3], RAI-refractory DTCs are identified by: a) presence of malignant/metastatic tissue that does not ever concentrate RAI, b) presence of tumor tissue that loses the ability to concentrate RAI after previous evidence of RAI-avid disease, c) RAI uptake in some lesions but not in others, and d) metastatic disease that progresses despite significant concentration of RAI.
It was shown that
BRAFV600E mutated PTCs display a greater reduction in the expression of genes involved in iodine uptake and organification, namely sodium/iodide symporter
NIS (
SLC5A5), thyroperoxidase (
TPO) and pendrin (
SLC26A4), compared with tumors with other mutations or without known genetic alterations [
4‐
6]. In vitro studies showed that the activation of
BRAFV600E down-regulates the expression of NIS and reduces RAI uptake in thyroid cells. On the other hand, no difference either in
NIS or in other thyroid differentiation genes was found in PTC with or without
RET/PTC rearrangements [
7], in accordance with the notion that these tumors rarely progress to aggressive or undifferentiated carcinomas [
8].
Few studies are currently available on the molecular profile of RAI-R thyroid cancers, either well differentiated, poorly differentiated or anaplastic, showing that they are enriched with
BRAF mutations, whereas
RAS mutations are more represented in RAI-A metastatic DTCs [
9,
10]. Based on these in vitro and in vivo data, pilot studies and phase 2 trials demonstrated that two selective BRAF inhibitors, namely Vemurafenib and Dabrafenib, are able to stimulate radioiodine uptake in patients with metastatic
BRAFV600E-mutant RAI-R DTCs [
11‐
16]. In addition, attempts of reverse RAI refractoriness in TC patients restoring radioiodine uptake, have been conducted with MEK inhibitors, tested as single agent [
17] or, more recently, in combination with BRAF inhibitors [
18,
19].
Even though the aberrant expression and/or function of NIS has been identified as factor for the lack of sensitivity to RAI, the mechanisms responsible for RAI resistance are still poorly understood. To get more insights into this topic, we investigated both the molecular profiles of RAI-A and RAI-R PTC patients, and their gene/miRNA expression. Indeed, though some studies have already described miRNA deregulation in PTC suggesting their possible role as diagnostic and prognostic markers [
20‐
24], any report is currently available about miRNA expression in relation to the response to RAI.
Therefore, aim of this study was to find possible molecular tags able to identify the avidity or refractoriness to radioiodine of PTCs, since this information is expected to have a major impact on the selection of the more appropriate treatment and follow up [
25].
Discussion
In this study we investigated the molecular profiles and gene/miRNA expression in metastatic PTC patients stratified for RAI response in accordance with their RAI-refractoriness/avidity.
By the PTC-MA platform we found a different molecular profile in RAI-avid and RAI-refractory PTCs.
BRAFV600E was significantly more frequent in PTCs unable to uptake RAI at the metastatic site already at the time of the diagnosis. An association between
BRAF mutation and RAI-R disease has been previously reported [
4‐
6,
9], though a whole molecular profile was not obtained and cases were not classified according to the “type” of RAI refractoriness, evident at the time of initial treatment or developed during time. Our data indicate that
BRAFV600E associates with an intrinsic tumor RAI refractoriness, being significantly more prevalent in tumors RAI−/D+. On the contrary, we report for the first time a higher prevalence of fusion genes in PTCs with initial RAI uptake, but without therapeutic efficacy (RAI+/D+), suggesting that also these molecular alterations interfere with the iodine organification process, though not impairing iodine uptake. Accordingly, the partial response recently reported in an advanced DTC harboring an
EML4-NTRK3 fusion and treated with Larotrectinib, a NTRK inhibitor, is consistent with the restoration of radioiodine organification [
44].
pTERT mutations were more frequent in the PTC group without RAI-uptake, in accordance with data that report for this genetic alteration a negative prognostic role in thyroid cancer [
45]. On the other hand, cases wild type for all the tested mutations and gene fusions were more prevalent in patients with RAI-avid metastatic disease in remission, indicating that gene alterations not analyzed by our assay are more frequently associated with a more differentiated and less aggressive disease.
In patients with available multiple tumor specimens, we found in most cases concordant genetic patterns between primary PTC and lymph node metastases, not only for
BRAFV600E mutation, in agreement with previous reports [
9], but also for
RET/PTC fusions. This was observed not only in synchronous LNMs but also in RAI-refractory LNM collected after RAI treatment.
The mutational profiles of primary and synchronous metastases in thyroid cancers, including PTC, have been already investigated [
46‐
50], describing high and significant concordance between the primary tumor and the corresponding metastasis, especially the LNM. On the other hand, only few studies assessed the global gene/miRNA expression in these tissue types [
43,
51,
52], confirming transcriptome similarity between primary TC and matched synchronous LNMs. To our knowledge, this is the first study investigating gene/miRNA expression in PTC tissues including RAI-refractory LNMs collected following RAI treatment.
We initially investigated a series including primary and LN metastatic PTCs, from either RAI-refractory or -avid patients. The gene profiles identified a clear separation between neoplastic and normal tissues, and tumor samples were also found to stratify into 2 major clusters, according to the tissues composition, and in particular to presence or not of an associated autoimmune thyroiditis or other types of immune cells infiltrating the tissue for primary tumors and NTs, and, of residual lymphoid tissue not replaced by neoplastic cells for LNMs. Indeed, PTC tissues including very high or low amount of immune cells displayed a distinguishable transcriptomic profile, which should be further evaluated for possible clinical impact in larger series.
On the other hand, the presence of these tumor infiltrating cells, of various origin, might have affected our analyses impairing a clear identification of PTC-specific mRNAs and miRNAs, as already reported [
31,
43,
53‐
55].
With the aim of identifying tumor related features, that could be not perturbed by the presence of other microenvironment derived cellular components, we thus focused on the subset of samples displaying high tissue purity from RAI-refractory patients.
In this sample set gene profiles confirmed not only the separation between tumors and NTs, but also and more interestingly, the transcriptome similarity among BRAFV600E mutated samples, either primary or LNMs post RAI, thus suggesting that radioiodine treatment minimally affects the expression profiles of RAI-refractory lymph metastases with respect to primary tumors.
In this samples group we also confirmed the up- and down- regulation of previously reported PTC-related miRNAs (revised in [
24]). In addition, we found a correlation with genotype and a more pronounced miRNA downregulation in
BRAFV600E and BRAF-like tumors, suggestive of more dedifferentiated tissues.
To the best of our knowledge this is the first study investigating gene/miRNA expression in tumor samples from RAI refractory patients, and including not only primary PTCs, but also synchronous and RAI-refractory LNMs. We found gene and miRNA deregulations consistent with those described by previous reports, such as the TCGA study [
30], which analyzed primary PTCs, regardless of the RAI classification. In addition, according with TCGA [
30], that describes how PTC subclassification is influenced by BRAF−/RAS-subtype and by the expression of thyroid related genes (TD score), along with the histological variant and driving lesion, we found that in our series from RAI-refractory patients (RAI+/D+ and RAI−/D+) the major classifying features are still the driving lesion (and in particular
BRAFV600E), the BRAF−/RAS-subtype and the TD score.
Among TD genes,
NIS was found to be significantly dowregulated in RAI-refractory tumors, and particularly in RAI−/D+ cases, consistent with the clinical behavior. As an original finding, also
SLC26A4 was significantly more downregulated in RAI−/D+ than in RAI+/D+ cases, suggesting that these cases are even more dedifferentiated and that if a small amount of RAI is uptaken, it is immediately washed out due to the lack of the apical transporter [
56]. Finally,
TPO was downregulated in both RAI refractory classes, as expected for tissues not able to organify and thus retain iodine into the colloid. By evaluating these crucial genes in relation to the genetic pattern, we found that
NIS appeared to be similarly reduced in
BRAFV600E and fusions positive tumors, whereas
TPO,
SLC26A4 and TD score were significantly more downregulated in
BRAFV600E tumors than in fusion positive tumors. Interestingly, these data were further confirmed by analyzing other series from public repositories and are consistent with the already reported dedifferentiation [
30] and RAI refractoriness associated with
BRAFV600E mutation [
9,
10].
Further studies to get more insights into the functional relationships among
BRAF and
RET/PTC oncogenes, RAI uptake and expression of thyroid differentiation genes such as
NIS and
SLC26A4, would be useful. In this context it was demonstrated that in mouse thyroid cancers with conditional
BRAFV600E expression the lack of radioiodine incorporation depend on BRAF activity [
57]. In the clinical setting, promising data on redifferentiation have been obtained with new compounds targeting BRAF or MEK, such as Vemurafenib, Dabrafenib, Trametinib, and Selumetinib, though the anti-tumor effect is variable, ranging from a partial to a null response, possibly due to the histopathological and genetic heterogeneity of mutated tumors [
11‐
19]. Finally, based on the original data obtained in the present work, future studies will be particularly focused on the possible effects of
RET/PTC oncogenes on RAI uptake, since to the best of our knowledge no data are available on this topic.
Conclusions
We studied the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory LNMs in correlation to RAI avidity or refractoriness.
BRAFV600E tumors were found more frequently unable to uptake RAI at the metastatic site already at the time of the diagnosis, suggesting an intrinsic RAI refractoriness. Accordingly, the genes related to RAI refractoriness, e.g. those responsible for iodine apical transport or organification, were highly downregulated, regardless of either the different tissue type (i.e. primary tumor, or synchronous or RAI refractory lymph nodal metastases) or the collection before or after RAI treatment, indicating that the dedifferentiation related to this mutation is present at the very early stages of tumor growth. Moreover, a transcriptome similarity was found among BRAFV600E mutated samples, either primary or lymph nodal metastases post RAI, thus suggesting that radioiodine treatment minimally affects the expression profiles of RAI-refractory lymph metastases with respect to primary tumors.
On the other hand, genes related to RAI refractoriness were less downregulated in fusions positive tumors which were, consistently, more frequently able to uptake RAI, but still with persistent disease, suggesting different mechanisms leading to RAI-refractoriness.
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