Molecular Diagnosis in Clinical Management and Diagnosis of Thyroid Cancer

Understanding the molecular landscape of thyroid cancer has advanced progressively with the current genomic developments in thyroid cancer, which has allowed us to discover novel targets for cancer management. The genetic modifications identified in the TCGA illustrate specific patterns mostly emphasizing two important signaling pathways namely mitogen-activated protein kinase signaling pathway (MAPK), and phosphatidylinositol-3-kinase (PI3K/AKT) signaling pathways ¹⁶. The genetic deregulations of transcription factors and kinases specific to such pathways lead to consequent activation that progresses and activates tumorigenic pathways and thereby progression of thyroid cancer. In the further sections, we will be discussing molecular modification in thyroid cancer.

The MAPK-signaling pathway is important for cellular development, differentiation, and progression. The pathway is commenced on the cellular surface by mitogenic stimuli which is a growth factor, that binds to tyrosine kinase receptor and phosphorylates the mitogen-activated protein kinase, and thereby downstream regulates the extracellular signal-regulated kinase (ERK). Phosphorylated signal-regulated kinase leads to the activation of transcription factors targeting genes for inducing cell cycle entry and suppressing negative regulation of the cell cycle ⁶. The MAPK-signaling pathway is regulated in various steps by enzymes and factors, for epigenetic modification can lead to subsequent active mechanisms, uncontrollable growth, and cellular proliferation, marking tumorigenesis. However, alterations in vital indicators of the MAPK-signaling pathway are present in almost 70% of all thyroid cancers.

A single point variation at T1799A in codon 600 in the BRAF oncogene, encoding serine and threonine kinase in the MAPK signaling pathway, causes BRAF (V600E) mutation ¹⁰. This mutation leads to active BRAF kinase which is independent of its upstream targeted gene, RAS, further leading to increased ERK activation. However, the mutant BRAF (V600E) kinase is 500 times more active than its wild type, and its variation is considered one of the significant originating events in the tumorigenesis of thyroid cancer ¹³. In addition, the BRAF (V600E) mutation is also responsible for activating the nuclear kappa beta factor (NFκB) mechanism which is crucial for inflammatory processes and activated during tumorigenesis.

Moreover, the development of telomerase reverse transcriptase (TERT) promoter mutations in thyroid cancer, illustrates an important incident in the thyroid cancer field, and has expanded its role in modifying the impact of driver mutations such as BRAF. The discovered mechanism of TERT promoter variation is to develop de novo binding elements of the ETS-transcription family of factors including GABPA, which causes telomere progression ³. However, the ETS-transcription factors are activated by MAPK signaling pathways, which affect the concomitant BRAF (V600E) and TERT mutations, and are thereby associated with aggressive forms of thyroid cancer.

The PI3K/AKT signaling pathway mainly regulates processes such as apoptosis, cellular proliferation, cell cycle development, cellular integrity, and ATP regulation, and leads to AKT activation ³. One of the important targets of the AKT signaling pathway is the RAS gene, a G-protein anchored to the inner cellular surface and located upstream of BRAF kinase and activated while bound to GTP. The variation in the RAS gene downregulates GTPase and causes it to an active GTP-bound state. Mutations in the RAS gene can activate both MAPK and AKT signaling pathways due to its location inside the cellular surface and adjacent to tyrosine kinase receptors. However, it is observed that RAS variations generally activate AKT signaling pathways in thyroid tumorigenesis ³⁰. However, the RAS mutation is pre-malignant and other variations are required to cause tumorigenesis. Moreover, PTEN mutations activate the AKT signaling pathway, which is the molecular foundation for follicular thyroid cancer occurrence in Cowden disorder. Variations in the PI3K3CA gene are also common during follicular thyroid cancer but are inferiorly differentiated in thyroid cancer and anaplastic thyroid cancer occurrence ³⁰.

The genomic translocations could lead to tumorigenic chromosomal rearrangements. The most usual group of translocations in thyroid cancer is RET/PTC, discovered by ¹⁴. It should be noted that it does not include a single translocation but a combination of distinct translocations including multiple genes along with RET. For instance, the commonly occurring translocations are RET/PTC1 and RET/PTC3 which illustrate translocation between the RET gene and CCD6 gene and RET gene and NCOA4 gene, respectively. The RET/PTC translocations happen as a mixture of genetic recombination between the 3’ tyrosine kinase of RET and the 5’ partner gene due to nuclear spatial propinquity. As RET is a proto-oncogene, any recombination of mutation during activation could lead to a tumor. Any rearrangement in RET/PTC gene encodes oncogenic processes including tyrosine kinase receptors, causing activation of MAPK and PI3K-AKT processes. The NTRK1, ALK, and PPARG are recombinant oncogenes where oncogenic fusion modifies the transcription of targeted genes, predisposing for tumorigenesis in thyroid cancer.

Epigenetic alterations of the genome and genetic expression involve histone modifications, and DNA and miRNA methylation. Such epigenetic markers impact the central dogma of DNA transcription, RNA translation, and protein synthesis inside the cell. Epigenetic alterations are common during human oncology and are also observed in thyroid cancer ³⁸. Methylation of DNA of promoter positions generally leads to genetic transcription suppression. In microarray-based genome-wide DNA methylation reports of thyroid cancer, done by ³⁰. reveal that similarity to sequencing reports shows low-frequency mutation in parathyroid cancer along with low frequency of mutation in DNA methylation ²⁰. In contrast, anaplastic thyroid cancer displays a large frequency of DNA methylation modifications ten times higher than parathyroid cancer ⁵.

miRNA involvement in thyroid cancer has been reported in the literature (He et al., 2005). In addition, long non-coding RNA (lncRNA) involvement in thyroid cancer has recently been developed by various scientists. Deregulated lncRNAs are actively involved in the epigenetic modification of genetic transcription and are drawn in in the suppression of tumors and other tumorigenic functions ¹⁹.

Thyroid nodules are very common among adults, however, the cancer frequency among individuals with thyroid nodules is less. Accordingly, thyroid cancer screening is not recommended generally. Ultrasound is considered the initial strategy for detecting malignancy and suspected nodules are detected through Fine Needle Aspiration (FNA). The usual limitation of FNA is indeterminate thyroid nodules. Moreover, the malignancy frequency ranges between 10-30%, with pathological examination needed to gain final insights (Ha et al., 2018). The molecular imaging techniques can successfully contribute to improving the preoperative analysis of indeterminate thyroid nodules.

Scintigraphy of thyroids done using either pertechnetate or sodium iodine is the specific diagnostic method for detecting thyroid nodules functioning autonomously and eradicating malignancy with even lower TSH values. In addition, molecular imaging with FDG (fluorodeoxyglucose) might aid in distinguishing benign from malignant indeterminate thyroid nodules ¹².

The growing evidence showing the involvement of AKT pathways in thyroid cancer pathogenesis and escape from traditional targeted therapies causes a closer look into multiple components of this pathway including the mTOR (mammalian target of rapamycin) component which when inhibited led to cellular proliferation suppression.

Two important mTOR suppressors significant during clinical trial data have been approved by the FDA for clinical usage for other cancers namely Temsirolimus in advanced kidney cancer and Everolimus for advanced pancreatic, breast, and renal carcinoma (Populo et al., 2012). Given the clinical benefits of these drugs and certainly mTOR activation as a therapeutic target in thyroid cancer, such targets are being examined for advanced thyroid cancer. Initial outcomes of phase II thyroid cancer clinical trial give positive hope. For instance, the NCT01025453 clinical phase II trial assessed the effect of Temsirolimus with Sorafenib in 37 thyroid cancer patients. Out of the total 37 patients, 8 patients responded partially, 21 patients had stable disease and only one patient developed progression while the remaining 7 patients were unassessable. Another clinical phase II trial NCT01141309 evaluates the combination of Everolimus and Sorafenib in 41 advanced thyroid cancer patients. In this cohort, 21 patients showed partial response, 14 patients had stable disease, and 3 patients had progressive disease. The cohort showed a better response in terms of toxicity assessment when compared to Sorafenib monotherapy, specifically in the differentiated thyroid cancer group.

In addition, the abundance of AKT and mTOR deformities in anaplastic thyroid cancer spots this pathway as an effective target for such patients. NCT02289144 is a clinical trial objecting to evaluate the impact of Ribociclib (targeting retinoblastoma variation) and Everolimus in malignant anaplastic thyroid cancer patients. The basis for targeting retinoblastoma in such patients is the abundance of intact retinoblastoma in anaplastic thyroid cancer patients ¹. However, more clinical trial study outcomes are awaited to suggest novel treatment strategies for anaplastic thyroid cancer patients since retinoblastoma is not considered a therapeutic target in these patients.

The fine needle aspiration (FNA) method is recently the most stable diagnostic strategy for thyroid nodules and represents the definite diagnosis of benign and malignant lesions in most cases, whereas 10-40% of all FNA samples are detected as indeterminate for malignant tumors ¹⁵. Owing to the limited definite diagnosis, most patients with indeterminate tumors undergo surgical treatment, however, only 10-20% of surgically removed tumors are cancerous. The patients with indeterminate FNA cytology and malignant cancers are not efficiently treated, as most of them undergo thyroid lobectomy and thyroidectomy.

Molecular diagnosis of FNA samples may improve the precision of cytological diagnosis of thyroid cancers. The most studied accumulated variation is BRAF mutation which has been reported in prospective and retrospective studies ^{24; 32}. In addition to BRAF mutation, various studies have suggested the probability of the detection of RET, TRK, and RAS variations in thyroid samples. However, the largest diagnostic effect can be gained by examining FNA samples for a cohort of variations rather than for a single mutation. Recent literature reports have suggested the diagnostic utility of molecular diagnosis for a cohort of mutations including BRAF, RAS, RET, and PAX8. A study examined 470 successive FNA samples from thyroid cancer that were examined and generated 32 variations which were strong predictors of malignant diagnosis after surgery ³². This report suggested that examining the mutation panel was helpful in predicting thyroid nodules with indeterminate cytology. In this group, the positive mutational status had 100% precision for depicting malignancy risk while the negative mutation status depicted benign nodules. Moreover, such a study represented that molecular examination reduced the false-negative frequency of cytology from 2-0.8%. in addition to BRAF mutation, RAS mutation also appeared to be of higher diagnostic value among FNA samples, as it depicted an 87-100% probability of malignancy. Interestingly, the RAS variations were determined in tumors, which are hard to diagnose cytologically alone, that is variants of papillary and follicular carcinoma. The diagnostic utilization of molecular markers has been reported effectively in Management Guidelines for patients with thyroid nodules and differentiated thyroid cancer, by the American Thyroid Association ¹¹. These guidelines suggested that using molecular markers including BRAF, RAS, RET, and PAX8 for elucidating FNA cytology helps in guiding patient care and management.

The radioactive iodine treatment for thyroid cancer in patients has been applied since 1946. Currently, a combination of therapy and diagnosis using radiolabelled somatostatin analogs has proven to be effective for managing somatostatin receptor tumors. The Lu¹⁷⁷-labelled somatostatin analogs binding receptors are the most promising radioactive therapy in clinical practice. Radioactive therapy might be considered as a potential therapy for differentiated thyroid cancer treatment with high expression of somatostatin receptor owing to efficient safety features and therapeutic value.

The association between thyroid cancer and the immune system has been explored largely owing to the concomitance of papillary thyroid cancer and Hashimoto thyroiditis. The dysregulation in the immune system and response partially correlates with differentiated thyroid cancer oncogenesis and development involving activation of immunosuppressors such as tumor-connected macrophages, immune checkpoints such as programmed death ligand-1 (PD-L1), and cytotoxic associate T-lymphocyte-4 (CTLA-4). PD-L1 was actively expressed in 50% of papillary thyroid cancers ³. This percentage increased to 61% in differentiated thyroid cancer and 70% in advanced papillary thyroid cancer, and 75% in anaplastic thyroid cancer ⁴. On the basis of these outcomes, the immunotherapeutic strategies involving immune checkpoint inhibitors may manage thyroid cancer effectively. The immune checkpoint inhibitors involve two major classes: those targeting CTLA-4 namely tremelimumab and ipilimumab, and targeting PD-L1 namely pembrolizumab, nivolumab, and atezolizumab. The immune checkpoint inhibitors augment the effector T cells reduce the regulation of suppressor cells and establish immune surveillance from which malignant cells can easily be removed. However, reports on immune checkpoint inhibitors for treating radioactive iodine refractory-differentiated thyroid cancer are still limited.

Conventionally, total thyroidectomy was performed in most anaplastic thyroid cancer patients, even though the American Thyroid Association standards endorse lobectomy for individuals with intra-thyroid low-risk differentiated thyroid cancer ¹⁸. Cervical lymph nodes occur in 20-50% of patients with differentiated thyroid cancer, and neck surgery dissection decreases the risk of tumor relapse. Prophylactic neck segmentation may progress control for invasive tumors, but it is dejected for reduced-risk differentiated thyroid cancer because potential morbidities are not justified by a significant clinical advantage. Pre-operative neck ultrasound generally suffices for planning surgery; however, supplementary cross-sectional imaging and magnetic resonance imaging are reserved for patients with locally advanced disease or for those who are at higher risk of establishing distant metastases. CT scan with [¹⁸F] FDG could be performed pre-operatively in aggressive differentiated thyroid cancer and anaplastic thyroid cancer. [25]. retrospectively analyzed 60 patients with lower-risk differentiated thyroid cancer who underwent CT scans before thyroidectomy. The authors reported very low sensitivity (10%) in elucidating lymph node metastasis with high specificity (90%) [25]. Few other studies compared the precision of positron emission tomography with that of neck ultrasound, and CT scans. It was observed that the specificities of all positron emission tomography, ultrasound, and CT scans are very high in evaluating both central and lateral neck regions. Although, the sensitivities are low (<50%) for all three methods. The total diagnostic precision of [¹⁸F] FDG positron emission tomography tends to be higher for lateral in comparison to central lymph nodes [9].

The total thyroidectomy followed by ¹³¹I management has continued to achieve an efficient diagnosis in multiple differentiated thyroid cancer patients. The ¹³¹I therapy for differentiated thyroid cancer is based on the principle of sodium iodide symporter articulating thyroid cells with differentiated thyroid cancer having the capability of confining circulating ¹³¹I. after surgical treatment, the risk of structural disorder relapse and persistence is evaluated using three-layer stratification (low, medium, and high) as recommended by the American Thyroid Association ¹⁸. The objective of therapeutic ¹³¹I administration after total thyroidectomy is delineated based on the standard guidelines as follows ².

1.Adjuvant treatment for irradiating suspected but neoplastic cell sites in low and medium-risk patients, as elucidated by histopathological features, thus decreasing disease relapse.

2.Treating known diseases for managing recurrent disorders in patients with determined metastatic disease.

3.Remnant excision to remove normal thyroid tissue fragments in low-risk patients, hence safeguarding minimal triglyceride levels, and facilitating follow-up.

Generally, two strategies for ¹³¹I treatment planning and delivery are approved in clinical practices: a method based on the clinical and pathological features and institutional procedures, and a strategy integrating post-operative radioiodine functional imaging. However, no pieces of evidence are available to permit recommending a single method over others and choice is based on local factors, including clinician and patient preferences.

Over the last few decades, understanding the underlying molecular pathways for thyroid differentiation and determination of important causal genes have led to the development of multiple novel radionuclide imaging strategies. A few variations such as EIF1AX and IDH1 and other signaling processes involving the differentiation process are still unclear, the significance of which needs to be explored. Various reports suggested that processes such as acetylation, glycosylation, methylation, and ubiquitination are closely associated with epigenetic oncogenesis ²⁸. There are also reports available in the literature suggesting proteomic evaluation in thyroid oncogenesis ³⁷. However, the underlying molecular mechanisms are still unknown. Moreover, clinical analysis of functional imaging of differentiated thyroid cancer has represented the importance of disease management and diagnosis, and further studies relevant to molecular targeted therapies are needed for better diagnosis and therapeutics of thyroid cancer in the future.

The advent of novel targeted therapy has unquestionably delivered us with more treatment options for advanced thyroid cancer. The emergent outcomes of phase II trials of Temsirolimus and Everolimus are expected to provide new insights into treatment options for thyroid cancer. However, how to utilize these weapons is of clinical importance, as whether to use them at an early stage of treatment or at completion of the current standard treatment mode. The timing of initiation with novel agents is of utmost significance. More explorations and examinations are required to address these issues. In the future, properly designed clinical trial studies will help us to better understand the comparative efficiency of novel agents. It is of utmost significance to determine an effective sequential and combined treatment method for minimizing resistance to inactive drugs in long-term clinical practice for advanced thyroid cancer management.