Review Article

The RET Oncogene in Papillary Thyroid Carcinoma Jason D. Prescott, MD, PhD; and Martha A. Zeiger, MD

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, accounting for greater than 80% of cases. Surgical resection, with or without postoperative radioiodine therapy, remains the standard of care for patients with PTC, and the prognosis is generally excellent with appropriate treatment. Despite this, significant numbers of patients will not respond to maximal surgical and medical therapy and ultimately will die from the disease. This mortality reflects an incomplete understanding of the oncogenic mechanisms that initiate, drive, and promote PTC. Nonetheless, significant insights into the pathologic subcellular events underlying PTC have been discovered over the last 2 decades, and this remains an area of significant research interest. Chromosomal rearrangements resulting in the expression of fusion proteins that involve the rearranged during transfection (RET) proto-oncogene were the first oncogenic events to be identified in PTC. Members of this fusion protein family (the RET/PTC family) appear to play an oncogenic role in approximately 20% of PTCs. Herein, the authors review the current understanding of the clinicopathologic role of RET/PTC fusion proteins in PTC development and progression and the molecular mechanisms by which RET/PTCs exert their oncogenic effects on C 2015 American Cancer Society. the thyroid epithelium. Cancer 2015;121:2137-46. V KEYWORDS: oncogene, papillary thyroid cancer, pediatric thyroid cancer, radiation, rearranged during transfection.

INTRODUCTION Thyroid carcinoma is a significant clinical problem. Approximately 62,980 new thyroid cancers will be diagnosed in the United States in 2014, representing an increase of almost 41% relative to the estimated 44,670 new thyroid cancers diagnosed in 2010. Furthermore, approximately 1890 deaths will be attributed to thyroid cancer in the United States in 2014. Thyroid malignancy is 2.5-fold more common in women than in men; and, between 1993 and 2001, the age-adjusted incidence of thyroid malignancy in American women increased at a rate of 4.3% per year: more rapidly than any other cancer subtype monitored by the Surveillance, Epidemiology, and End Results (SEER) database.1 Papillary thyroid carcinoma (PTC) accounts for the vast majority of thyroid cancers and is defined by its distinctive cytologic and histologic features. Characteristic cytologic findings include large, crowded nuclei; small nucleoli; nuclear grooves/clefts; nuclear pseudoinclusions (which are thought to represent redundant nuclear membrane); and a nuclear clearing/ground-glass appearance that results from relatively open/euchromatic DNA organization. Histologic features of PTC include well defined papillae composed of 1 or 2 layers of relatively eosinophilic cancer cells surrounding a fibrovascular core (Fig. 1). Colloid, the storage form of thyroid hormone, is generally scant; and psammoma bodies, which are calcified, fibrotic condensations that may represent infarcted papillae, are observed in up to 80% of histologic specimens.2 Refinement in histologic evaluation has allowed further subtyping of some PTCs according to the presence or absence of particular additional microscopic features, and these features have been correlated with disease behavior and prognosis. Histologic subclassification is relevant in approximately 15% of PTCs, and subtypes include the follicular variant, tall cell, solid, hobnail, cribriform, diffuse sclerosing, insular, trabecular, oncocytic, microfollicular, clear cell, and pseudoWarthin categories. With the exception of the follicular variant, which generally portends an excellent prognosis, these subtypes are associated with more aggressive behavior, including increased rates of local invasion, locoregional lymph node spread, and distant metastases. Although several oncogenic mutations, including rearranged in transformation (RET)/PTC fusions, have been loosely correlated with some of these PTC subtypes, the precise molecular alterations that distinguish these variants from classic PTC remain to be defined.3,4

Corresponding author: Jason D. Prescott, MD, PhD, Assistant Professor of Surgery and of Oncology, Department of Surgery, The Johns Hopkins School of Medicine, 600 N. Wolfe Street/Blalock 605, Baltimore, MD 21287; Fax: (410) 502-1891; [email protected] Endocrine Surgery, Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland We thank Matthew T. Olsen, MD, and Justin A. Bishop, MD, for providing the cytologic and histologic images of papillary thyroid cancer used to produce Figure 1 in this article. DOI: 10.1002/cncr.29044, Received: July 8, 2014; Revised: August 21, 2014; Accepted: August 26, 2014, Published online March 2, 2015 in Wiley Online Library (wileyonlinelibrary.com)

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Figure 1. (A) Representative papillary thyroid carcinoma (PTC) cytology is observed, including prototypical nuclear crowding (yellow arrow) and nuclear clearing (red arrow; original magnification 340). (B) Representative PTC histology reveals the papillary structure characteristic of PTC. The dashed black line outlines single layers of tumor cells that define colloid-depleted fibrovascular papillae (original magnification 310).

Elucidation of the molecular underpinnings of PTC remains an area of significant research interest, and several specific oncogenic mutations appear to play a role in PTC development. Among these, it has been demonstrated that activating mutations involving the RET proto-oncogene have particular importance, and these mutations are the focus of the current review. The RET Proto-Oncogene

The RET proto-oncogene is located on the long arm of chromosome 10 (10q11.2) and encodes a 150-kD receptor tyrosine kinase.5 RET is relatively well conserved among species, and RET homologs have been identified in several vertebrates as well as in Drosophila melanogaster.6,7 RET is encoded by 21 exons, and several RET messenger RNA (mRNA) splice variants—each of which specifies a unique protein isoform with apparently distinct cellular functions—have been described.8,9 The RET protein is characterized by 3 separate functional domains: 1) an extracellular domain that contains 4 cadherin-like repeats, potentially mediating receptor oligomerization, and a cysteine-rich region likely involved in intraprotein disulfide bond formation; 2) a transmembrane domain; and 3) an intracellular domain that includes multiple tyrosine residues necessary for RET protein kinase activity. The RET extracellular domain undergoes glycosylation in the endoplasmic reticulum, ultimately producing the mature 170-kD RET protein.10,11 RET gene expression is present in most neuronal cell populations, including enteric, sensory, sympathetic, and motor neurons. RET is expressed in urogenital and neural crest cells during vertebrate development, in which it plays a crucial role in renal morphogenesis, spermatogenesis, 2138

and formation of the nervous system. RET gene expression is absent, or is present at only very low levels, in normal thyroid epithelium, and normal thyroid development does not require RET expression.12-14 Activation of RET protein kinase activity is mediated through binding between the RET extracellular domain and a ligand-bound coreceptor protein from the glial cell-derived neurotrophic factor (GDNF)-family receptor a (GFRa) superfamily. Ligands known to bind GFRa proteins and, thus, to activate RET include persephin, artemin, and neurturin, all of which are members of the GDNF family (a subgroup of the transforming growth factor b [TGFb] superfamily).15 Binding between RET and a ligand-bound GFRa protein results in autophosphorylation of multiple tyrosine residues within the intracellular enzymatic domain of the RET protein. The resulting changes in RET protein conformation allow binding of 1 or more cytoplasmic adapter proteins, depending on the pattern of tyrosine residue phosphorylation present, which then mediate the activation of 1 or more cytoplasmic signaling cascades (Table 1). Adapter proteins known to interact with specific cytoplasmic RET phosphotyrosine residues include growth factor receptorbound protein 7 (Grb7)/Grb10/Grb14, Grb2, SRC proto-oncogene (src), src homology 2 domain-containing transforming protein 1 (shc), src homology 2 domaincontaining transforming protein C3 (ShcC), phospholipase C-c, fibroblast growth factor receptor substrate 2 (FRS2), insulin receptor substrate 1 (IRS1), IRS2, PDZ and LIM domain 5 protein (Enigma family protein), docking protein 1 (Dok1), Dok4/Dok5, Dok6, SH3 and multiple ankyrin repeat domains 3 (Shank3), and protein kinase Ca (for a review, see Arighi et al16). The identities Cancer

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RET Mutations in Papillary Thyroid Ca/Prescott and Zeiger

TABLE 1. Adapter Proteins Known to Interact With Specific C-Terminal RET Protein Phosphotyrosine Residues and Their Associated Downstream Intracellular Targets Adapter Protein

RET C-Terminal Phosphotyrosine for Adapter Dockinga

Major Intracellular Target/Target Pathway

Grb2 Grb7/Grb10/Grb14 Phospholipase C-g Src Shc ShcC Shank3 FRS2 IRS1/2 Enigma Dok1 Dok4/5 Dok6 PKCa STAT3

Y905, Y981, Y1015, Y1096 Y429, Y905, Y981, Y1015, Y1096 Y539, Y905, Y981, Y1015, Y1096 Y905, Y981, Y1015, Y1096 Y1062, Y586 Y1062 Y1062 Y1062 Y1062 Y1062, Y586 Y1062 Y1062 Y1062 Y1062 Y752, Y928

PI3K/AKT, ubiquitinization PI3K/AKT MAPK PI3K/AKT MAPK, PI3K/AKT, ubiquitinization PI3K/AKT MAPK, PI3K/AKT MAPK, PI3K/AKT PI3K/AKT Ubiquitinization MAPK MAPK ? ? Induction of gene expression: VEGF, cyclin D1, intracellular adhesion molecule 1 Unknown

Unknown

Y687, Y826, Y1029

Reference(s) Hennige 2000,17 Scott 200518 Balogh 201219 Lundgren 201220 Encinas 200121 Hayashi 200022 Pelicci 200223 Schuetz 200424 Melillo 200125 Melillo 200126 Kales 200227 Murakami 200228 Uchida 200629 — — Hwang 200330 —

Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; Dok6, docking protein 6; Enigma, PDZ and LIM domain 5 protein (Enigma family protein); FRS2, fibroblast growth factor receptor substrate 2; Grb, growth factor receptor-bound protein; IRS1/2, insulin receptor substrates 1 and 2; PI3K, phosphatidylinositol-3-kinase; PKCa, protein kinase Ca; RET, rearranged in transformation; Shc, homology 2 domain-containing transforming protein 1; ShcC, src homology 2 domain-containing transforming protein C3; Src, SRC proto-oncogene; STAT, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor. a See Romei and Elisei, 2012.16

of downstream cellular pathways modulated in response to activated RET-adapter protein interactions are complicated and have only begun to be defined. Nonetheless, several of these pathways have been implicated in cell proliferation, migration, chemotaxis, differentiation, and survival. In particular, phosphorylation of RET cytoplasmic tyrosine 1096 is necessary for docking of the shc intracellular adapter protein, which can then recruit Grb2/ GRB2-associated binding protein (GAB) to activate the phosphatidylinositol-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/Akt/mTOR) cell survival/apoptosis pathway, or Grb2/son of sevenless (Sos), resulting in activation of the mitogenic mitogen-activated protein kinase (MAPK) cascade (Fig. 2).22,32 RET gene mutations have been linked to the development of multiple human diseases. Loss of RET function is associated with the development of Hirschprung disease, for example; and it is known that several distinct RET-activating point mutations induce c-cell transformation, producing medullary thyroid cancer (MTC). It is noteworthy that the clinical behavior of RET-mediated MTC depends on the specific mutation involved. For example, a germ-line RET missense mutation resulting in a methionine to threonine substitution at position 918 within the RET protein produces aggressive disease, characterized by early local invasion and metastasis, whereas Cancer

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replacement of serine by alanine at position 891 produces MTC that is far less likely to spread from the thyroid (for review, see Arighi et al16). Although their molecular underpinnings remain to be characterized, these reliable MTC genotype-phenotype relations have allowed for the development of clinical management guidelines that are based on the specific RET mutation present. In addition to their oncogenic roles in MTC, such germ-line RET mutations are also responsible for syndromic tumor development in other endocrine organs (classified as multiple endocrine neoplasia type 2A or 2B, depending on the endocrine tissues involved). Although the underlying molecular mechanisms involved remain unknown, both the pattern of endocrine organ involvement and the penetrance of associated tumor formation depend on the specific RET mutation present. In addition to their oncogenic activity in the development of MTC, it has been demonstrated that RET mutations also induce PTC. In contrast to other RETinduced malignancies, however, which depend on activating point mutations within the endogenously expressed RET gene, the mutational mechanism whereby RET mediates PTC involves chromosomal translocation. Invariably, these translocations produce hybrid proteins in which the oncogenic RET carboxy terminal kinase domain is fused to the amino terminal end of an unrelated gene, the expression of which remains dependent on its 2139

Review Article

Figure 2. This chart illustrates the organization of rearranged during transformation (RET) kinase domain tyrosine residues known to undergo phosphorylation and the associated, respective adapter proteins and downstream pathways that are activated. Tyrosine residues known to undergo phosphorylation are indicated in red. Associated adapter proteins are shown in boxes. Arrows indicate the respective targeted pathways. AKT indicates v-akt murine thymoma viral oncogene homolog; Dok1, docking protein 1; Dok4/5, docking proteins 4 and 5; Dok6, docking protein 6; EC, extracellular domain; Enigma, PDZ and LIM domain 5 protein; FRS2, fibroblast growth factor receptor substrate 2; Grb2, growth factor receptor-bound protein 6; Grb7/10/14, growth factor receptor-bound proteins 7, 10, and 14; IRS1/2, insulin receptor substrates 1 and 2; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3 kinase; PKCa, protein kinase Ca; Shank3, SH3 and multiple ankyrin repeat domains 3; Shc, src homology 2 domain-containing transforming protein 1; ShcC, src homology 2 domain-containing transforming protein C3; Src, SRC protooncogene; STAT3, signal transducer and activator of transcription 3; TM, transmembrane domain; VEGF, vascular endothelial growth factor.

own endogenous promoter. The molecular underpinnings of oncogenesis mediated by these distinct mutational mechanisms, which may have significant molecular and clinical implications, have yet to be characterized. The Molecular Functions of RET/PTC Fusions in PTC

The RET oncogene was discovered in 1985 through experiments involving lymphoma DNA extracts, which, when transfected into NIH3T3 mouse fibroblast cells, produced malignant transformation.33 Characterization of the resulting purified transforming element revealed a fusion protein in which the RET carboxy terminal kinase domain was constitutively active and oncogenic. Subsequent analysis of human tumors revealed that oncogenic RET fusion proteins, termed RET/PTCs, were a particular feature of approximately 20% of PTCs.34 To date, 13 different oncogenic RET/PTC fusion proteins (termed RET/PTC1-PTC9) have been discovered. These include pericentriolar material 1 (PCM1)RET; ret finger protein (RFP)-RET; hook protein 3 (HOOK3)-RET; and rich in glutamic acid (E), leucine (L), lysine (K), and serine (S) (ELKS)-RET (Fig. 3) (for a review, see Arighi et al16). Each chimera is produced by a distinct chromosomal translocation event in which the 2140

promoter and 50 region of a heterologous gene encoding a thyrocyte-expressed protein are fused, in frame, to the kinase-encoding 30 end of the RET proto-oncogene. RET/ PTC1 accounts for approximately 60% of RET-associated PTC, with RET/PTC3 representing approximately 30% and RET/PTC2 representing 10%. The remaining RET/ PTC family members are extremely rare. For reasons that are not understood, the chromosomal break point involved in each tumor always occurs in intron 11 of the RET proto-oncogene. The involved 50 -fusion component is not related to the basic oncogenic function of the fusion protein in each tumor, as long as the relevant promoter specifies thyrocyte expression (RET is otherwise not expressed in the thyroid epithelium) and the encoded amino terminal protein fragment mediates dimerization. RET/PTC-induced thyrocyte transformation depends on constitutive activation of the RET protein kinase, which, in turn, requires fusion protein dimerization. This dimerization is mediated by the amino-terminal fusion component, and functional analysis of these components confirms the presence of 1 or more probable dimerization domains in each tumor. The specific molecular events promoted by constitutive RET kinase domain activity that mediate thyrocyte oncogenesis are still under investigation. Up-regulation of Cancer

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Figure 3. The known members of the rearranged in transformation/papillary thyroid carcinoma (RET/PTC) fusion protein family are shown. Each known 50 fusion gene partner fragment is depicted on the left, and the 30 RET kinase domain fusion component is depicted on the right. ARA70 indicates androgen receptor-associated protein 70; ELE1, RET-activing gene ELE1 (nuclear receptor coactivator 4); ELKS, rich in glutamic acid (E), leucine (L), lysine (K), and serine (S); H4/ CCD6, cell division control protein 6; HOOK-3, hook protein 3; hTIFg, human transcriptional intermediary factor 1g; KTN1, kinectin 1; PCM-1, pericentriolar material 1; PRKAR1A, cyclic adenosine monophosphate-dependent protein kinase type 1a regulatory subunit; PTC1, papillary thyroid carcinoma 1; RFG, RET-fused gene; TM, transmembrane domain; TRIM24, tripartite motif-containing 24; TRIM27, tripartite motif-containing 27; TRIM33, tripartite motif-containing 33.

several different genes, including the p27kip1 cyclindependent kinase inhibitor gene, the osteopontin cytokine gene (and its receptor) and the chemokine (C-X-C motif) receptor 4 (CXCR4) gene have been implicated in RET/ PTC-mediated thyrocyte oncogenesis.35-37 Inhibition of microRNA miR-199a-3p, with resultant overexpression of the mesenchymal epithelial transition factor (MET) and mammalian target of rapamycin (mTOR) genes, may also play a role in RET/PTC-induced thyroid malignancy.38 RET/PTC can also preferentially activate the mitogenic epidermal growth factor receptor, which, in turn, may contribute to thyrocyte transformation through direct activation of the downstream MAPK pathway.39 Finally, analysis of genomic methylation patterns from primary human tissues also reveals relative hypermethylation in RET/PTC-associated thyroid tumors, suggesting significant oncogenic differences in gene expression patterns between malignant and benign thyroid tissues.40 Characterization of the normal cellular function of each respective donor gene that contributes to an amino terminal end of a RET/PTC fusion protein is variable (Table 2). Several lines of evidence suggest that disruption of some of these genes, resulting from the translocation Cancer

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event necessary to produce a RET/PTC fusion, could synergize with RET/PTC during thyrocyte oncogenesis. The cell division control protein 6 (H4/CCD6) gene, which contributes its 50 end to RET/PTC1, participates in the regulation of programmed cell death. H4/CCD6 overexpression can induce thyrocyte apoptosis, whereas inhibition of H4/CCD6 expression appears to promote cell survival. In addition, expression of a truncated carboxyterminal H4/CCD6 protein may act as a dominant, negative inhibitor of the wild-type H4/CCD6 protein, thus inhibiting apoptosis.41 Similarly, the protein kinase A regulatory subunit 1-a (PRKAR1A) gene and the tripartite motif-containing (TRIM) family genes, which contribute 50 ends to the RET/PTC2, RET/PTC6, RET/PTC7, and RFP-RET fusions, respectively, normally act as cancer suppressors, a function almost certainly abrogated by their disruption during fusion gene generation.42,43 In addition, TRIM proteins exhibit oncogenic function in some contexts.44 Finally, the specific subcellular localization of individual RET/PTC fusion proteins likely depends on information specified by their respective amino terminal fusion components. Resultant differences in the intracellular localization of RET/PTCs may modulate their oncogenic activity. This may explain, for example, why some studies suggest differences in the clinical aggressiveness of RET/PTC1-derived PTCs relative to those associated with the RET/PTC3 fusion.49 RET/PTCs in Radiation-Induced PTC

Exposure to ionizing radiation, especially during childhood or adolescence, is the only significant modifiable risk factor associated with the development of PTC. Long-term follow-up studies of atomic bomb survivors, victims of nuclear accidents, and patients who received radiotherapy for various medical conditions reveal an increase in PTC incidence beginning approximately 5 years after initial exposure. This increase has been associated with exposures of as little as 0.1 grays, and the magnitude of PTC risk increases linearly with increasing exposure dose.50 Radiation-associated PTC, in particular, is associated with RET/PTC fusion protein expression. Assessments of surgically resected PTCs from patients who lived in proximity to the Chernobyl nuclear facility during its containment breach reveal the presence of RET/PTC fusions in up to 72% of analyzed tumors compared with 20% of PTCs harboring RET/PTC fusions in nonirradiated populations.51 This finding is unsurprising, because ionizing radiation produces double-stranded breaks in DNA; and it is believed that the generation of aberrant 2141

Review Article TABLE 2. 50 RET/PTC Donor Genes 50 RET/PTC Donor Gene H4/CCD6 PRKAR1A ELE1/ARA70/RFG ELE1/ARA70/RFG RFG5 TRIM24/hTIF TRIM33/hTIFc KTN1 RFG9 ELKS PCM-1 TRIM27/RFP HOOK-3

Hypothesized Normal Function

Chromosomal Locus

RET/PTC Oncogene

Regulation of apoptosis Cancer suppressor Androgen/steroid receptor signaling Androgen/steroid receptor signaling Unknown Transcription factor/second messenger/E3 ubiquitin ligase Histone modification/E3 ubiquitin ligase Endoplasmic reticulum/cytoskeleton organization Unknown Synaptic factor Centriole organization

10q21 17q23 10q11.2 10q11.2 14q 7q32-34

RET/PTC1 RET/PTC2 RET/PTC3 RET/PTC4 RET/PTC5 RET/PTC6

1p13 14q22.1 18q21-22 12p13.3 8p21-22

RET/PTC7 RET/PTC8 RET/PTC9 ELKS-RET PCM1-RET

6p21

RFP-RET

8p11.21

HOOK3-RET

Reference Celetti 200441 Sandrini 200242 Alen 199943 Alen 199943 — Hatakeyama 201144 (review) Hatakeyama 201144 (review) Lin 201245 (review) — Hida & Ohtuska 201046 (review) Avidor-Reiss & Gopalakrishnan 201347 (review) Hatakeyama 201144 (review) Shotland 2003

48

Transcription factor/second messenger/E3 ubiquitin ligase Intracellular trafficking

Abbreviations: ARA70, androgen receptor associated protein 70; ELE1, RET-activating gene ELE1 (nuclear receptor coactivator 4); ELKS, rich in glutamic acid (E), leucine (L), lysine (K), and serine (S); H4/CCD6, cell division control protein 6; HOOK-3, hook protein 3; hTIFc, human transcriptional intermediary factor 1g; KTN1, kinectin 1; PCM-1, pericentriolar material 1; PRKAR1A, cyclic adenosine monophosphate-dependent protein kinase type 1a regulatory subunit; PTC, papillary thyroid cancer; RET, rearranged in transformation; RFG, RET-fused gene; RFG5, RET-fused gene 5 protein; RFG9, RET-fused gene 9 protein; RFP, ret finger protein; TRIM24, tripartite motif-containing 24; TRIM27, tripartite motif-containing 27; TRIM33, tripartite motif-containing 33.

chromosomal translocations, including those that produce RET/PTC fusion genes, depends on this type of DNA damage. The relation between ionizing radiation exposure and RET/PTC-mediated PTC is further supported by the observation that only the RET/PTC1 and RET/PTC3 fusions are present with high frequency among radiation-associated PTCs. The RET protooncogene localizes to the long arm of chromosome 10 (10q11.2), as do the 50 donor genes that contribute to the RET/PTC1 and RET/PTC3 fusions (H4/CCD6 and RETactiving gene ELE1/androgen receptor-associated protein 70/RET-fused gene [ELE1/ARA70/RFG], respectively). It is believed that this contiguous physical correlation between fusion partners dramatically increases their physical proximity, such that a single ionizing radiation path may produce both double-stranded DNA breaks necessary for RET/PTC fusion gene generation. Translocation by paracentric inversion then occurs at high frequency to produce the RET/PTC1 and RET/PTC3 fusion oncogenes. This hypothesis is supported by studies demonstrating that the RET and H4/CCD6 genes are directly juxtaposed in 35% of normal human thyrocytes during interphase, whereas juxtaposition is present in only 6% of normal interphase mammary epithelial cells, thus making RET/PTC fusion unlikely in these cells.52 This observation also explains why other RET/PTC family fusions, for which contributing genes are not physically juxtaposed, are rare. Finally, studies examining DNA radiation sensitivities have revealed a particular predisposition of the 2142

RET gene to radiation-induced fragmentation, and it has been demonstrated that ionizing radiation exposure produces RET/PTC fusions both in cultured cells and in mouse xenografts.53-55 Several observational and experimental findings strongly support an oncogenic role for RET/PTC fusions in the initiation of PTC. For example, RET/PTC oncogenes are highly prevalent in presumably nascent PTCs, including microcarcinomas and occult disease.56 In addition, the expression of an RET/PTC fusion in human primary thyroid epithelial cell cultures produces cytologic features consistent with PTC.57 RET/PTC fusion expression can also transform cultured rat thyrocytes, whereas thyroid-specific expression of RET/PTC1 and of RET/ PTC3 in transgenic mice results in the development of thyroid tumors that histologically resemble PTC.58-60 Nonetheless, RET/PTC gene fusions do not always produce PTC. Although RET/PTC-mediated thyroid oncogenesis is specific for PTC, a growing body of evidence indicates that RET/PTC fusions also are expressed in benign thyroid disease, including thyroid adenomas and Hashimoto thyroiditis.61,62 Nonclonal RET/PTC expression appears to correlate with benign disease, whereas clonal thyrocyte RET/PTC expression is specific for PTC. These findings suggest that the cellular microenvironment also plays an important role in promoting thyroid carcinogenesis, even in the context of RET/PTC expression. This theory is supported by studies demonstrating accelerated growth, but not actual malignant Cancer

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behavior, of thyroid nodules that harbor subpopulations of cells in which nonclonal RET/PTC is detected.63 It is important to note that any interpretation of the relation between RET/PTC oncogenes and thyroid disease depends on the accurate detection of RET/PTC expression. Unfortunately, there are limitations associated with each method used to assay for RET/PTC fusion (reviewed by Nikiforov34). Karyotyping cannot detect RET/PTC3, requires growing cells, and does not verify protein expression. Southern blot analysis cannot distinguish clonal from nonclonal RET/PTC cell distributions and, like karyotyping, cannot assess for the expression of identified RET/PTC fusions at the protein level. Florescence in situ hybridization does not distinguish between RET/PTC subtypes and also cannot verify fusion protein expression. Reverse transcriptase-polymerase chain reaction analysis carries a high false-positive rate and cannot detect novel RET/PTC fusions. In situ hybridization and immunohistochemistry cannot differentiate RET/PTC fusions from wild-type RET RNA or protein, respectively. Indeed, differences in detection methodology likely explain the wide differences in RET/PTC incidence reported for PTC by some investigators.64,65 This picture is further complicated by the finding that more than 1 RET/PTC family oncogene may be present in a single tumor.66 The Role of RET/PTC Fusions in Pediatric PTC and in PTC Behavior

There is a clear correlation between patient age and the probability that a given PTC will harbor an RET/PTC fusion. One study identified RET/PTC fusions in 67% of PTCs from patients who ranged in age from 4 to 19 years, whereas such fusions were present in only 32% of tumors originating in adults ages 31 to 80 years.67 Given the correlation between RET/PTC rearrangement and exposure to ionizing radiation, this finding may be in part because of an increased susceptibility of pediatric patients to radiation-induced DNA damage. RET/PTC1 and RET/PTC3 fusions account for the vast majority of RET/PTC-positive PTCs in pediatric patients, and some investigators have reported correlations between the particular RET/PTC family member expressed and disease behavior. Studies that examined PTC development among children who were exposed to ionizing radiation after the Chernobyl nuclear disaster revealed differential latency between radiation exposure and tumor development, with RET/PTC3 tumors identified preferentially within 10 years of exposure and RET/ PTC1 fusions occurring primarily in tumors that developed greater than 10 years later.68 Relative tumor aggresCancer

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siveness has also been associated with different RET/PTC family members, although these findings remain controversial. RET/PTC fusions are less common in the indolent follicular variant of PTC relative to other histologic subtypes. Furthermore, expression of the RET/PTC3 fusion has been associated with the relatively aggressive solid histologic PTC variant, whereas RET/PTC1 expression has been linked to the more indolent classic variant.69,70 These histologic correlations have been confirmed in transgenic mouse experiments, although findings refuting these data have also been reported.71 Local disease recurrence and the development of distant metastases in pediatric patients have also been preferentially associated with RET/PTC3 relative to RET/PTC1, and it has been observed that transgenic mice harboring the RET/PTC3 mutation develop far more aggressive PTC than their RET/PTC1-mutant counterparts. RET/PTC in Thyroid Nodule Diagnosis and in PTC Treatment

The development and application of diagnostic and therapeutic techniques that exploit specific molecular features underlying human diseases are of paramount importance in clinical medicine. The preoperative diagnosis of PTC, in particular, remains a markedly inaccurate process in many patients; therefore, new preoperative testing methods intended to improve diagnostic accuracy by assessing mutational profiles of thyroid nodules have recently been developed.72,73 However, as detailed above, RET/PTC rearrangements are not specific to PTC when their cellular distributions are nonclonal. Furthermore, an optimized method for accurately detecting the presence of all 13 known RET/PTC fusions has yet to be developed. Thus, the preoperative detection of RET/PTC rearrangements in thyroid needle aspirates has not proven useful in optimizing the surgical management of thyroid nodules.74 The majority of PTCs are effectively treated by thyroidectomy, with or without postoperative radioiodine therapy. Nonetheless, a significant proportion of PTCs will be unresectable and residual/recurrent postsurgical disease, and/or distantly metastatic PTC, may be refractory to radioiodine therapy. Great interest has therefore developed in generating novel therapeutic agents that target those specific molecular changes specifying oncogenic behavior in these tumors. Several such agents have been discovered that are capable of inhibiting RET protein kinase activity as well as the enzymatic activities of other tyrosine kinase family members (Table 3).75 Indeed, clinical trials have been initiated to assess the safety and efficacy of many of these new medications in the management of 2143

Review Article TABLE 3. Small-Molecule Inhibitors of RET Kinase Activity Inhibitor

Kinases Inhibited

Phase Study Completed

Vandetanib

RET, VEGFR, EGFR

Phase 2 for treatment-refractory PTC Phase 2 for treatment-refractory PTC Phase 3 for medullary carcinoma Phase 3 for treatment-refractory PTC Phase 3 for treatment-refractory PTC None, preclinical studies only

Sunitinib Cabozantinib

RET, VEGFR, PDGFR RET, VEGFR2, MET, MTC

Lenvatinib Sorafinib PP1 and PP2

RET, KIT, VEGFR1-2, FGFR, PDGFRb RET, Raf kinase, VEGFR, PDGFR RET, SRC

Abbreviations: EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; KIT, tyrosine-protein kinase Kit; MET, mesenchymal epithelial transition factor; MTC, medullary thyroid carcinoma; PDGFR, platelet-derived growth factor receptor; PDGFRb, platelet-derived growth factor receptor b; PP1, protein phosphatase 1; PP2, protein phosphatase 2; PTC, papillary thyroid carcinoma; Raf kinase, rapidly accelerated fibrosarcoma serine/threonine-specific protein kinase; RET, rearranged in transformation; SRC, SRC proto-oncogene, nonreceptor tyrosine kinase; VEGFR2, vascular endothelial growth factor receptor 2.

treatment-refractory PTC, although clinical studies specifically assessing the role of such agents in the treatment of RET/PTC-mediated cancer remain to be performed. Conclusions

The RET/PTC family of oncogenes, in particular RET/ PTC1 and RET/PTC3, plays a key role in the development of some PTCs, especially pediatric PTCs and those associated with ionizing radiation exposure. Moreover, the relative aggressiveness of a given PTC may depend on the specific RET/PTC family member expressed, suggesting that important differences in intracellular behavior exist among these oncoproteins. Finally, the nonclonal expression of RET/PTC oncogenes observed in some benign thyroid diseases indicates that the local cellular microenvironment may play a role in modulating RET/ PTC oncogenic activity. Together, these observations reveal significant deficits in the current understanding of the mechanisms whereby RET/PTC mediates thyroid tumorigenesis, mechanisms that, when characterized, may provide pivotal insights into the development of new PTC therapeutics. The fields of molecular-based diagnostics and molecule-targeted therapeutics are both in their infancy; and, perhaps not unexpectedly, simple preoperative screening for the presence of RET/PTC fusions in thyroid nodule aspirates has not proven to be useful in refining surgical decision making. This finding may be secondary to imperfect RET/PTC detection methodology and/or an incomplete understanding of the mechanisms whereby 2144

RET/PTC mediates tumorigenesis. Improvements in RET/PTC detection techniques and in our understanding of the specific molecular events whereby RET/PTC fusions produce carcinoma may dramatically improve the clinical utility of preoperative RET/PTC oncogene screening. In addition, relatively nonspecific targeting of cellular tyrosine kinases using novel agents has produced some promising therapeutic results. Research in this area will certainly continue, ultimately producing progressively more efficacious and less toxic therapeutics for otherwise treatment-refractory PTC. FUNDING SUPPORT No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures.

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