Figure 43-1 Schematic illustration of the key intracellular signaling pathways involved in the pathogenesis of differentiated thyroid carcinoma.
Figure 43-2 Schematic illustration of the key intracellular signaling pathways involved in the pathogenesis of medullary thyroid carcinoma.
A second group of chromosomal translocations associated with PTC comprise mutations in the NTRK gene that lead to formation of various TRK oncogenes. Similar to RET/PTC, these fusion TRK proteins combine the N terminus of one of several genes normally expressed in thyroid follicular cells with the tyrosine kinase domain of the receptor for nerve growth factor. 9 These mutations are seen in about 5% of PTC.
These chromosomal rearrangements involving tyrosine receptor genes are commonly seen following exposure to ionizing radiation and may be the leading mechanism of thyroid oncogenesis in this setting. This has been particularly true following the unfortunate experiences of the Chernobyl nuclear power plant accident as well as atomic bomb explosions in Japan. 10,11 Spatial proximity of RET or NTRK genes to the heterologous donors of the N-terminal promoter regions specific to chromosomal folding in thyroid follicular cells may permit a single radiation event to cause double-strand breaks in each gene, thus permitting the recombination event.
Other RTKs have been found to be overexpressed in DTC cells and may be relevant to disease biology. In addition to EGFR, these include platelet-derived growth factor receptor (PDGFR) α and β; vascular endothelial growth factor receptors (VEGFR) 1 and 2; fibroblast growth factor receptors (FGFR); and hepatocyte growth factor receptor (MET). Genetic abnormalities are rarely observed, and epigenetic alterations may be contributory; however, the exact mechanisms leading to overexpression are generally not known.
BRAF
Mutations of the serine-threonine kinase BRAF are the most common oncogenic abnormality reported in PTC. 13 Activated frequently in other cancers as well, BRAF has high affinity for binding and phosphorylating MEK isoforms in the MAPK pathway. Although point mutations in multiple codons have been reported to cause constitutive activation of BRAF, the most studied and most frequent is a valine-to-glutamine substitution at amino acid residue 600 (denoted V600E mutation). By destabilizing the inactive conformation of the kinase, the V600E BRAF mutation causes the protein to remain in a catalytically competent conformation that allows continuous phosphorylation of MEK. BRAF mutations are virtually never seen in benign thyroid lesions, and thus the presence of a BRAF mutation can be pathognomonic of a malignancy if detected in a cytologically suspicious biopsy specimen. 15 Of note, a rare translocation mutation of BRAF (causing fusion of the AKAp9 gene with BRAF) has been reported to cause PTC after radiation exposure.
BRAF mutations occur in 40% to 50% of cases of PTC (especially the classical and tall-cell variants) and are also frequent in PDTC and ATC. Multiple studies describe a more aggressive phenotype associated with these mutations, including higher rates of lymph node metastases, extrathyroidal extension, poor radioiodine uptake and response to therapy, and advanced stage at presentation. Prognostically, BRAF mutations are associated with higher rates of recurrence and worse survival. 13,16 In a model of conditional activation of the V600E BRAF mutant in thyroid follicular cells, mice develop rapidly growing poorly differentiated tumors with negligible expression of thyroid-specific genes such as the sodium-iodide symporter as well as loss of iodine incorporation; these changes are reversible on inhibition of BRAF or MEK kinase functions. 17 Other downstream effects of mutant BRAF include alterations in DNA methylation and increased expression of genes associated with invasive and metastatic disease such as matrix metalloproteinases. 18
RAS
Point mutations in RAS genes are among the most common oncogenic abnormalities in all cancers, and DTC is no different. Mutations in the RAS protein lead to constitutive activation through alterations in the binding affinity of the kinase for GTP or through inactivation of its intrinsic GTPase activity. Thus, mutant RAS can signal downstream through both the MAPK and PI3K/Akt pathways without upstream activation derived from ligand-bound RTK. All three RAS genes (H-RAS, K-RAS, and N-RAS) are implicated in thyroid tumor formation from follicular cells, including 20% to 40% of benign follicular adenomas, 40% to 50% of FTC (including 15% to 20% of oxyphilic variants), 10% to 20% of PTC (almost exclusively follicular variants of PTC), and 25% of PDTC. 19–22 The presence of a RAS mutation may portend more aggressive disease with worse outcomes, but this has not been extensively examined. 19,23 Each of these histologies has also been observed in transgenic mice expressing RAS mutations, although the presence of mutant RAS proteins alone is likely insufficient to cause tumor formation. 24,25
PI3K/Akt Pathway
Inactivating germline mutations of the tumor suppressor gene PTEN cause Cowden syndrome, which carries a 50- to 70-fold increased risk for the development of DTC, especially FTC. 26,27 Loss of this tumor suppressor function leads to activation of PI3K, Akt, and mTOR, thus contributing to enhanced cell cycle progression, decreased apoptosis, and increased tumor proliferation. However, mutations in individual genes in this pathway are otherwise uncommonly reported as early oncogenic events. Instead, somatic mutations and/or overexpression of PIK3CA (which encodes the class I p110α catalytic subunit of PI3K), AKT, and PTEN are observed as frequent later events, especially in FTC, PDTC, and ATC. 20,28,29 Gene amplification as well as activating point mutations are observed in 10% to 20% of PDTC and 40% of ATC and can be found in tumors also bearing either BRAF or RAS mutations. AKT activation is also characteristic of the invasive fronts of aggressive DTC and has been reported to trigger increased cellular motility. 30
PAX8/PPARγ
A chromosomal translocation, t(2:3) (q13;p25), results in the PAX8/PPARγ mutation, which couples the DNA binding domains of the thyroid transcription factor PAX8 with the entire coding sequence of the nuclear peroxisome proliferator-activated receptor subtype γ1. 31 The actual mechanisms by which the encoded fusion protein contributes to thyroid tumorigenesis remain unclear. However, several critical pathways may be affected, including reduced expression of PTEN leading to increased activation of Akt, and a dominant-negative effect on the normal PPARγ transcription factor permitting enhanced cellular proliferation and reduction of apoptosis. 30,32 This mutation may be preferentially seen in younger patients with smaller tumors, which are generally better prognostic signs, but conversely are also seen in tumors with solid or nested histologies as well as with vascular invasion. 33
Medullary Thyroid Carcinoma
RET
About 20% of MTC occurs in one of several familial syndromes: multiple endocrine neoplasia (MEN) 2A (which also includes parathyroid tumors and pheochromocytomas); MEN 2B (which also includes pheochromocytomas, intestinal ganglioneuromatosis, neuromas of the tongue and subconjunctiva, and Marfanoid habitus); and familial MTC (FMTC, which lacks the other clinical features of MEN 2A). Additional variants of MEN 2A have been reported that include cutaneous lichen amyloidosis and with Hirschsprung disease. Germline mutations in RET were identified as causative of these hereditary forms of MTC in two landmark 1993 studies. 34,35 Today, more than 99% of all cases of hereditary MTC can be attributed to one of numerous point mutations in RET that cause activation of the tyrosine kinase function of the RTK (Table 43-1 ). Given the ubiquitous nature of the mutation, it is not surprising that the disease begins with diffuse hyperplasia of all of the C cells, with eventual development of one or more malignant foci.
The most common germline mutation, a cysteine-to-arginine substitution at codon 634 (denoted C634R), accounts for at least half of all cases of MEN 2A and has also been extensively studied in vitro in the well-characterized TT cell line. 36 This mutation is found in the cysteine-rich extracellular domain of RET, a region responsible for ligand-dependent dimerization. However, in the setting of the C634R mutation, RET is capable of ligand-independent dimerization, leading to autophosphorylation of the intracellular tyrosine residues that are responsible for interaction with downstream signaling pathways. In contrast, a methionine-to-threonine substitution at codon 918 (denoted M918T) is associated with the more aggressive phenotype of MEN 2B. The M918T mutation occurs in the intracellular domain of RET, changing the conformation of the tyrosine kinase domain and allowing marked enhancement of autophosphorylation in the absence of dimerization. In addition, allelic imbalance, due to either increased copy number of the mutant RET allele or deletion of part or all of the wild-type allele, has been reported in several cases of MEN 2A as well as the TT cell line itself.
Table 43-1
Most Common Mutations of the RET Gene Causing Hereditary Medullary Thyroid Carcinoma
CLA, Cutaneous lichen amyloidosis; FMTC, familial medullary thyroid carcinoma; HD, Hirschsprung disease; MEN 2A, multiple endocrine neoplasia type 2A; MEN 2B, multiple endocrine neoplasia type 2B.
From Hu MI, Jimenez C, Cote G, et al. Medullary thyroid carcinoma. In: Braverman LE, Cooper DS, eds. Werner & Ingbar’s The Thyroid: A Fundamental and Clinical Text. 10th ed. Philadelphia, Pa: Wolters Kluwer; 2013:744-764.
Sporadic MTC, on the other hand, is not associated with germline changes in RET, but nonetheless, somatic RET mutations have been commonly reported in 25% to 50% of sporadic MTC cases. In this instance, the most frequent somatic mutation is the M918T alteration, but numerous other codon changes have also been observed, including selected deletions as well as point mutations. Of note, about 6% to 7% of patients with clinically sporadic MTC are found to carry germline mutations diagnostic of hereditary forms of the disease despite the absence of a positive family history, thus leading to the consensus recommendation to recommend RET germline testing for all newly diagnosed cases of apparently sporadic MTC. 37,38
Extensive genotype:phenotype correlations have been established in the two decades since RET was identified as causing MTC. In addition to identifying specific clinical syndromes associated with each mutation, these analyses have also demonstrated that disease penetrance, typical age of development of C-cell hyperplasia and malignancy, and the aggressiveness of the malignancy vary in a manner that is based to a large degree on the individual mutation. Thus, the intracellular domain mutations, which tend to be associated with the aggressive MTC characteristic of MEN 2B, are also found to cause aggressive sporadic MTC when they occur as somatic mutations. Patients who present with sporadic MTC associated with a somatic M918T mutation of RET have worse outcomes, including overall survival. 39 These genotype:phenotype correlations are also useful in determining the role and outcomes of genetic screening in hereditary disease. Recently published guidelines from the American Thyroid Association divide known RET germline mutations into four risk categories that guide earliest age for RET testing of potential familial carriers, earliest age for recommended first thyroid ultrasound and serum calcitonin testing to detect early presymptomatic evidence of disease, and role for potentially curative prophylactic thyroidectomy. 38 Using this type of approach, most young patients identified by prospective genetic screening as carriers for FMTC or MEN 2A can be cured with prophylactic thyroidectomy, although a small percentage remain with biochemical evidence of residual disease. 40
RAS
Mutations of RAS have recently been recognized as common in sporadic MTC in the absence of documented RET mutations. 41,42 A wide range of frequency has been reported, however, between 10% and 80% of all RET–wild-type sporadic cases, using differing techniques for identifying RAS mutations. In the largest study, tumor samples from 108 sporadic disease patients without somatic RET mutations were subjected to RAS sequencing, yielding a frequency of 17% in that setting. 42 Of the three potential genotype combinations, patients who were (mutant)RAS (wt)RET were more likely to be disease free after a median follow-up of 5 years than those who were (wt)RAS (wt)RET or (wt)RAS (mutant)RET.
Other Molecular Mechanisms Active in Thyroid Carcinoma
As described earlier, tumor cells in DTC often express or overexpress cell surface RTKs for a variety of circulating growth factors, including VEGFR, FGFR, EGFR, PDGFRβ, IGFR, and MET. In addition, MTC cells also can contain similar cell surface RTKs for growth factors, including EGFR, MET, and FGFR. Overall, their roles appear to enhance the proliferative effects of mutated RTKs and intracellular signaling kinases, but in certain settings they may have critical functions. For example, studies of cancer stem cells, such as those derived from the MTC cell line MZ-CRC-1 that contains the M918T RET mutation, demonstrate the dependence on FGFR in the presence of RET knockdown for continued sphere formation and stem-cell proliferation. 43
Of clear importance, however, is the role of growth factors secreted by thyroid tumor cells that interact with the neighboring stromal cells. This is particularly relevant for angiogenesis, by which tumor cells stimulate growth of vascular structures for supply of nutrition and oxygen as well as a conduit for distant metastasis. Cells from both DTC and MTC actively secrete various VEGF isoforms under conditions of limited oxygen, particularly VEGF-A, which interacts with VEGFR on neighboring vascular endothelial cells. 44–47 Similarly, cells from both DTC and MTC generate hepatocyte growth factor and FGF to stimulate MET and FGF receptors, respectively, on neighboring cells, an important pathway for stimulating angiogenesis in the absence of VEGFR activity. 48–50
The TSH receptor has an indispensable role to stimulate thyrocyte proliferation normally. In neoplastic cells, the receptor is generally expressed and functional in DTC and PDTC, but usually absent in ATC. Short-term increases in the level of TSH can stimulate malignant cells with a functioning receptor, as evidenced by increases in differentiated functions such as thyroglobulin production and radioiodine incorporation, and tumor proliferation is similarly observed from chronic exposure to high levels of TSH. 51 There has also been recent evidence to suggest that TSH is necessary for BRAF-induced thyroid carcinogenesis. 52
The tumor suppressor gene TP53 is frequently mutated in advanced thyroid cancers. Point mutations that inactivate the suppressor protein are often seen in PDTC and ATC but are not seen in DTC. 53,54 Reexpression of normal p53 protein restores differentiated function in vivo. 55 Combining TP53 mutation with other genetic lesions in animal models can reproduce the phenotype of ATC. 56,57
The Wnt/β-catenin signaling pathway is often activated in advanced thyroid cancers, but recent data suggest a possible early role as well. Mutations in the scaffold proteins APC and Axin, along with β-catenin itself, have been reported in a majority of PDTC and ATC tumors, associated with increased tumor proliferation and loss of tumor differentiation. 58 In RET/PTC-mutant PTC, increased β-catenin localized to the nucleus has been observed as a result of posttranslational modification and protein stabilization. 59 Patients with familial adenomatosis and Gardner’s syndrome, associated with mutations in the APC gene, have high risk for development of PTC, particularly an aggressive cribriform-morular variant. 60 In cases that have been examined, the presence of a germline APC mutation is associated with very high expression of β-catenin in the PTC cells along with frequent mutations in the gene as well as in RET/PTC. 61
Therapeutic Targeting
The development of tyrosine kinase inhibitors (TKIs) that target many of the key oncogenes and other molecular abnormalities in thyroid cancer has led to the investigation of many agents in treatment of patients with advanced and metastatic disease. 62 Initial efforts focused on drugs that could inhibit activated RET kinase, such as vandetanib. 63 In this early study, a tyrosine kinase inhibitor that primarily inhibited VEGFR and EGFR was shown to block autophosphorylation of M918T and RET/PTC3, to prevent growth of cell lines with RET/PTC1 mutations, and to inhibit growth of tumors after injection of fibroblasts transformed with the RET/PTC3 gene. Based on findings like these, numerous multitargeted TKIs that can inhibit both RET and VEGFR (as well as other kinases) have been studied in the laboratory and in clinical trials. For example, vandetanib and cabozantinib (which also inhibits MET) significantly improve progression-free survival in patients with metastatic MTC. 64,65 Both drugs appear to be slightly more effective in patients whose tumors have RET mutations, but remain beneficial even in the absence of the targeted mutation. It remains to be determined whether these agents primarily work by targeting mutant RET kinase, through inhibition of angiogenesis through VEGFR (and MET for cabozantinib), or other mechanisms such as inhibiting the normal function of wild-type RET.
In DTC, initial interest in RET inhibitors waned when it was recognized that RET/PTC mutations are uncommon in advanced and metastatic disease. Instead, focus has been placed on targeting VEGFR-mediated angiogenesis with multikinase inhibitors such as motesanib, sorafenib, and sunitinib. 66–69 More recently, the availability of highly selective inhibitors of individual kinases in the MAPK pathway such as vemurafenib, dabrafenib, and selumetinib, has enabled trials evaluating oncogene targeting. Phase I experience with the two BRAF inhibitors, vemurafenib and dabrafenib, suggests that about one third of patients with BRAF-mutant PTC may respond to therapy 70,71 ; phase II studies are under way. Selumetinib, an MEK inhibitor, has been studied in a fascinating pilot trial of 20 patients with progressive, radioiodine-refractory PTC, in which a 5-week course of selumetinib therapy followed immediately by radioiodine scanning induced restoration of enough radioiodine uptake and retention to permit high-dose radioiodine therapy to be subsequently administered to 7 of the patients; partial responses were observed in 5 patients who received the radioiodine therapy. 72
Moving beyond inhibitors that target single pathways or kinases, studies are now under way to evaluate rational combinations of agents. For example, preclinical studies suggest that simultaneous inhibition of both MAPK and PI3K pathway signaling in DTC and MTC may be more effective than inhibiting either pathway individually, providing the rationale for combining drugs such as sorafenib and everolimus. Similarly, inhibition of both BRAF and MEK in melanoma yielded a potentially more effective regimen with fewer side effects than use of a BRAF inhibitor alone, and this approach is also being tested in BRAF-mutant PTC. 73
Future Directions
The molecular abnormalities described here represent a broad effort during the past 20 years to understand the fundamental pathophysiology of thyroid cancer. Whereas early oncogenic events have been identified that probably account for the majority of these tumors, further study is needed to identify the incipient events in the remaining tumors. Just as critical is the need for a more comprehensive understanding of the steps that lead to progression, invasion, metastasis, and occasional dedifferentiation. An integrated framework will be required that merges knowledge of DNA mutations with understanding of the role of epigenetic alterations, changes in miRNA regulation of gene expression, and other fundamental processes that contribute to the malignant phenotype. Clinical trials of therapies to reverse genetic changes and alter complex signaling abnormalities will need to be informed by comprehensive, individualized tumor profiling that will facilitate the selection of the correct combination of therapies for each individual patient, including the identification of patients with sufficiently indolent tumors that no therapy will ever be required.
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