Pathogenesis of Thyroid Carcinoma

Massimo Santoro and Francesca Carlomagno

Abstract
This chapter summarizes our current knowledge about molecular lesions driving
the most common subtypes of thyroid carcinoma. Genetic lesions in the RET
receptor tyrosine kinase and in RAS family GTPases are present in a large
proportion of sporadic medullary thyroid carcinomas (MTC). RAS mutations
are also common in well-differentiated thyroid carcinomas of the papillary (PTC)
and follicular (FTC) type. Genetic lesions, most commonly the V600E point
mutation, in the BRAF serine/threonine kinase are common in PTC; the PAX8-
PPARG gene fusion is present in FTC. Finally, aggressive thyroid cancer types
are enriched in several additional mutations including those targeting the TP53
tumor suppressor and the TERT (telomerase-reverse transcriptase) gene pro-
moter. This knowledge is being translated into novel diagnostic and prognostic
markers as well as molecular targets for novel therapeutic options.

Keywords
Kinase • Neoplastic progression • Differentiation • MAPK • TP53 • TERT

Abbreviations
4E-BP1 4E-binding protein 1
AKT AKR mice tyhymoma oncogene
ALK Anaplastic lymphoma kinase
APC Adenomatous polyposis coli
ARID AT-rich interaction domain
ART Artemin
ATC Anaplastic thyroid carcinoma

M. Santoro (*) • F. Carlomagno

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II,
Naples, Italy
e-mail: masantor@unina.it

# Springer International Publishing AG 2016 1

P. Vitti, L. Hegedus (eds.), Thyroid Diseases, Endocrinology 3,
DOI 10.1007/978-3-319-29195-6_20-1
2 M. Santoro and F. Carlomagno

ATRX ATP-dependent helicase X

BRAF B-type rapidly accelerated fibrosarcoma oncogene
CCDC6 Coiled coil domain containing protein 6
CEA Carcinoembryonic antigen
CLA Cutaneous lichen amyloidosis
CLD Cadherin like domain
CRD Cysteine rich domain
CREBBP Cyclic AMP responsive element binding binding protein
CTNNB1 Catenin beta 1
CV-PTC Classical variant-papillary thyroid carcinoma
DDR DNA damage response
DICER1 Dicer 1 ribonuclease III
DTC Differentiated thyroid carcinoma
EIF1AX Eukaryotic translation initiation factor 1A, X-linked
ERK Extracellular regulated kinase
ETS E-twenty six
FAP Familial adenomatous polyposis of colon
FNMTC Familial non medullary thyroid carcinoma
FOXE1 Forkhead box E1
FTA Follicular thyroid adenoma
FTC Follicular thyroid carcinoma
FTEN Familial thyroid epithelial neoplasia
FV-PTC Follicular variant-papillary thyroid carcinoma
GABP GA binding protein
GAP GTPase activating protein
GDNF Glial cell-derived neurotrophic factor
GEF Guanine nucleotide exchange factor
GTP Guanosine triphosphate
HCC Hürthle cell carcinoma
HD Hirschsprung's disease
KD Kinase domain
KMT2A/C/D Lysine methyltransferase 2 A/C/D
MAPK Mitogen-activated protein kinase
MEN Multiple endocrine neoplasia syndrome
MLH1 Mut L homolog 1
MLH3 Mut L homolog 3
MMR Mismatch repair
MNG1 Multinodular goiter 1
MSH2 Mut S homolog 2
MSH6 Mut S homolog 6
MTC Medullary thyroid carcinoma
mTOR mammalian target of rapamycin
mTORC mammalian target of rapamycin complex
NCOA4 Nuclear coactivator 4
NF1 Neurofibromatosis 1
Pathogenesis of Thyroid Carcinoma 3

NF2 Neurofibromatosis 2
NIFTP Non invasive follicular thyroid neoplasm with papillary like
nuclear features
NKX2-1 NK2 homeobox 1
NMTC Non-medullary thyroid carcinoma
NMTC1 Non-medullary thyroid carcinoma 1
NRT Neurturin
NTRK Neurotrophic tyrosine kinase
PAX8 Paired box gene 8
PBMR1 Polybromo-1, BRG1-associated factor
PDK1 Phosphoinositide-dependent kinase 1
PDTC Poorly differentiated thyroid carcinoma
PI3K Phosphatidylinositol 3 kinase
PI3KCA Phosphatidylinositol 3 kinase catalytic subunit
PIP3 Phosphatidylinositol (3,4,5)-trisphosphate
PKB Protein kinase B
PPARG Peroxisome proliferator-activated receptor gamma
PPFP PAX8-PPARG fusion protein
PSP Persephin
PTC Papillary thyroid carcinoma
PTEN Phosphatase and tensin homolog
RAS Rat sarcoma oncogene
RBD Ras binding domain
RET Rearranged during transfection
RSK Ribosomal protein S6 kinase
RTK Receptor tyrosine kinase
SBS Single base substitution
SETD2 SET domain containing 2 protein
SMARCB1 SWI/SNF related matrix associated actin dependent regulator of
chromatin B1
SOS Son of sevenless
SWI/SNF SwItch/sucrose non-fermentable
TC Thyroid carcinoma
TCGA The cancer genome atlas
TCO Thyroid tumors with cell oxyphilia
TCV-PTC Tall cell variant-papillary thyroid carcinoma
TERT Telomerase reverse transcriptase
THADA Thyroid adenoma associated protein
TP53 Tumor protein p53
TSC2 Tuberous sclerosis 2
TSH Thyroid stimulating hormone
WNT Wingless-related integration site
YAP Yes-associated protein
4 M. Santoro and F. Carlomagno

Contents
Epidemiology of Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Classification of Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Etiology of Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Molecular Pathogenesis of Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Molecular Lesions in Papillary Thyroid Carcinoma (PTC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Molecular Lesions in Follicular Thyroid Carcinoma (FTC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Molecular Lesions in Anaplastic (ATC) and Poorly Differentiated (PDTC)
Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Molecular Lesions in Medullary Thyroid Carcinoma (MTC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Epidemiology of Thyroid Carcinoma

In 2015, the estimated incidence of thyroid carcinoma (TC) is 64,300 new cases in
the United States, rendering TC the fifth most common cancer type in women if
non-melanoma skin cancers are excluded (Cabanillas et al. 2016; Siegel et al. 2016).
TC incidence is highest in countries with a high human development index (HDI),
and it has been stably rising over the last 10 years. This increase is, at least in part,
related to increased surveillance and use of high-resolution diagnostic imaging
methods (Cabanillas et al. 2016; Siegel et al. 2016; Dralle et al. 2015; Fagin and
Wells 2016).

Classification of Thyroid Carcinoma

TC is classified based on the cell type of origin (C cells or follicular cells) and the
expression of differentiated features (Giordano 2016; Nikiforov and Nikiforova
2011). TC arising from neuroendocrine calcitonin-producing C cells is called med-
ullary thyroid carcinoma (MTC), while TC arising from endoderm-derived follicular
cells is called non-medullary thyroid carcinoma (NMTC). MTC accounts for
approximately 3–5% of all TC cases (Fagin and Wells 2016; Howlader et al. 2016;
Wells et al. 2013, 2015). NMTC is further subdivided in four major types (PTC,
FTC, PDTC, ATC), whose relative prevalence depends on geographic factors,
including iodine intake in the region of interest (Cabanillas et al. 2016; Fagin and
Wells 2016). Papillary (PTC) and follicular (FTC) TC, together with other rare types,
such as Hürthle cell (oncocytic) carcinomas (HCC), belong to the differentiated
thyroid carcinoma (DTC) category. PTC is the most common TC subtype overall
(Cabanillas et al. 2016; Fagin and Wells 2016). Poorly differentiated (PDTC) and
anaplastic (ATC) carcinomas are rare TC subtypes characterized by a partial or total
loss of differentiation, respectively. TC histological classification is strongly related
to disease outcome. DTC is generally associated with a favorable outcome and with
Pathogenesis of Thyroid Carcinoma 5

Fusions in PTC.
Missense mutations of RET
(less commonly indels,
RET, NTRK1, fusions) in MTC.
NTRK3, ALK, Rare ALK fusions in MTC.
others

RTK

Missense mutations of
codons12, 13, 61 in MTC Missense mutations
(HRAS>KRAS), or FTC, in PDTC and ATC.
PTC, PDTC and ATC
(NRAS>HRAS>KRAS).

RAS PI3K
NRAS,
Missense or
HRAS, PTEN
PiP3 truncatinglesions in
KRAS
PDTC and ATC.
BRAF
PDK1
RAF AKT
V600E (less commonly
K601E, other
missense mutations,
indels , fusions) in Missense mutations
PTC, PDTC, ATC. in PDTC and ATC.
MEK mTOR

ERK

Fig. 1 Schematic representation of the ERK and PI3K signaling pathways. Next the two pathways,
individual proteins most commonly involved in thyroid cancer are indicated. Type(s) of genetic
lesions associated to the different types of thyroid cancer (MTC, PTC, FTC, PDTC, ATC) are
indicated in the red boxes

a long-term survival rate >90%, though up to 30% of patients may relapse after
initial treatment (Mazzaferri 1999; Liebner and Shah 2011; Xing et al. 2013). ATC
has a rapid onset and fulminant disease course with a mean survival of only 6 months
(Fagin and Wells 2016; Smallridge et al. 2009; Smallridge and Copland 2010).
PDTC has a behavior that is intermediate between DTC and ATC, with a mean
survival of approximately 3 years (Fagin and Wells 2016; Xing et al. 2013).

Etiology of Thyroid Carcinoma

Both environmental and genetic factors are relevant for the etiology of TC.
The incidence of FTC, in particular, and possibly ATC, is highest in regions with
iodine deficiency (Zimmermann and Galetti 2015). Mechanistically, iodine defi-
ciency can lead to cancer promotion secondary to chronic TSH stimulation due to
impaired thyroid hormones biosynthesis (Dumont et al. 2015). High iodine content
has also been related to increased TC incidence (Dralle et al. 2015). Exposure to
ionizing radiation, particularly in childhood, has been recognized as a risk factor for
PTC. Persons who have received external radiotherapy for the treatment of various
head and neck diseases, or have been exposed to radiation after the atomic bomb
6 M. Santoro and F. Carlomagno

explosions in Hiroshima and Nagasaki, or to radioisotopes after the Chernobyl

accident have shown increased PTC incidence (Williams 2008, 2015). Mechanisti-
cally, ionizing radiation can lead to oncogenic rearrangements, secondary to double-
strand DNA breaks, and illegitimate recombination of genes that map in spatial
proximity in the interphase chromatin of thyrocytes (Gandhi et al. 2010). Chromo-
some fragility may further contribute to these recombination events (Dillon et al.
2013). Exposure to environmental pollutants may also play a role in TC. Increased
TC incidence has been reported in volcanic areas, such as Hawaii, Iceland, and
Sicily, possibly due to inorganic elements present in pollution (Duntas and Doumas
2009; Malandrino et al. 2016). Accordingly, in the area of Mount Etna in Sicily, a
twofold increase in incidence of TC has been associated with increased levels of
several trace elements in the water and in the residents’ urine samples (Malandrino
et al. 2016).
About one quarter of MTC cases have a monogenic pattern of inheritance (see
below). Familial inheritance has also been described in up to 9% of the NMTC cases
(Fagin and Wells 2016; Mazeh and Sippel 2013; Navas-Carrillo et al. 2014).
Accordingly, first-degree relatives of TC patients carry a risk that is four to tenfold
higher than the general population (Hsiao and Nikiforov 2014). Familial NMTC can
either occur as one of the components of familial multicancer syndromes (familial
adenomatous polyposis of colon (FAP), Cowden’s disease, Carney complex,
Werner’s syndrome, McCune-Albright syndrome) or as a predominant feature (iso-
lated FNMTC) (Navas-Carrillo et al. 2014; Bonora et al. 2010). Most FNMTC are
PTC; some cases show specific histological features, such as trabecular struma with
oxyphilia or cribriform pattern in FAP patients (Mazeh and Sippel 2013; Navas-
Carrillo et al. 2014). Increased multifocality, invasion, lymph-node metastases, and
recurrence as well as early age at onset have been described in some FNMTC cases
with respect to sporadic counterparts. An autosomal dominant pattern of inheritance
with incomplete penetrance and variable expressivity has been documented in most
isolated FNMTC. However, FNMTC is a genetically heterogeneous condition, and
different candidate susceptibility loci have been involved in different families. These
include MNG1 (chr. 14q31), TCO (chr. 19p13.2), fPTC/PRN (chr. 1q21), NMTC1
(chr. 2q21), FTEN (chr. 8p23.1), NKX2–1 (chr. 14q13.3), and FOXE1 (chr.
9q22.33) (Mazeh and Sippel 2013; Navas-Carrillo et al. 2014; Gudmundsson et al.
2009). In one pedigree with multiple TC cases, a highly penetrant germline mutation
was found to target the DICER1 gene (chr. 14q31) whose protein product is involved
in microRNA processing (Rio Frio et al. 2011).

Molecular Pathogenesis of Thyroid Carcinoma

Currently, more than 90% of TC cases have at least one recognized pathogenetic
genetic lesion (Hsiao and Nikiforov 2014). Simplistically, most of the targeted genes
code for proteins that function along two particular signaling pathways: the ERK
(extracellular regulated kinase) and the PI3K (phosphatidylinositol 3-kinase)
Pathogenesis of Thyroid Carcinoma 7

PTC
unknown
RET, other RTKs
RAS
BRAF

TERT Histone MT
TERT PI3K/AKT MMR
PI3K/AKT EIF1AX SWI/SNF
EIF1AX TP53 NF1/NF2

PDTC ATC
unknown
unknown
RET and ALK
RAS
RAS
BRAF
BRAF

Fig. 2 Prevalence of the major genetic driver lesions in thyroid cancer. The prevalence of driver
mutations (BRAF, RAS, and RET or other RTKs) in papillary, poorly differentiated, and anaplastic
thyroid carcinomas is shown in the pie charts (Fagin and Wells 2016; Cancer Genome Atlas
Research Network 2014). In the case of PTC, lesions in RET and other RTKs (NTRK1, NTRK3,
ALK, MET, LTK, and FGFR2) are pooled together. Shown BRAF lesions include valine-to-
glutamate mutation at residue 600 (V600E) only. RAS lesions are single amino acid substitutions
of glycine 12, glycine 13, and glutamine 61 of HRAS, NRAS, and more rarely KRAS. Unknown
areas include samples without any identified driver lesion. Lesions that co-occur with the
abovementioned driver lesions in the progression to aggressive cancer subtypes (PDTC and ATC)
are indicated. TERT (telomerase-reverse transcriptase) lesions include promoter mutations; MMR
mismatch repair genes, histone MT histone methyltransferases. See text for additional details

signaling cascades (Mendoza et al. 2011). However, as discussed below, other

molecular circuits are involved in TC, as well.
The ERK pathway is one of several MAPK (mitogen-activated protein kinases)
signaling modules in eukaryotes (Dhillon et al. 2007). Its activation is typically
initiated by the binding of a growth factor to its cognate membrane receptor endowed
with tyrosine kinase catalytic activity (RTK) (Fig. 1). This is followed by the
activation of the kinase activity of the RTK and the recruitment of the guanine
nucleotide exchange factor (GEF) SOS, which mediates GTP nucleotide loading on
RAS small GTPases. Secondary to recruitment to GTP-bound RAS, serine/threonine
RAF kinases (ARAF, BRAF, CRAF) are activated through dimerization and phos-
phorylation (on serine 338 and other sites) and phosphorylate (on serine 217/serine
221) MEK proteins. MEKs, in turn, are dual (tyrosine and serine/threonine) activity
kinases which phosphorylate (on threonine 202/tyrosine 204) the serine/threonine
kinases p44 and p42 ERKs. Finally, activated ERKs, directly or via other down-
stream kinases such as p90RSK, potently affect cellular transcriptional output by
8 M. Santoro and F. Carlomagno

SP TM
CLD1 CLD2 CLD3 CLD4 CRD TKD1 TKD2
RET 100 aa

break M918T

PL S1 S2 HR
HRAS
KRAS 20 aa
NRAS
G12/13 Q61

RBD CRD
KD
BRAF 100 aa

break V600E

Fig. 3 Schematic representation of RET, RAS, and BRAF proteins and their most common genetic
lesions in thyroid cancer. RET: SP (signal peptide), CLD1 to 4 (cadherin-like domains 1 to 4), CRD
(cysteine-rich domain), TM (transmembrane domain), TKD1 and 2 (tyrosine kinase domain,
subdomain 1 and 2). The position of the breakpoint (break) of RET in RET/PTC fusions and the
most common RET point mutation in medullary thyroid carcinoma (methionine-to-threonine
mutation at residue 918) are indicated. RAS: PL (P loop, involved in binding the γ phosphate of
GTP), S1/2 (switch regions 1 and 2, which regulate binding to RAS regulators and effectors), HR
(hypervariable region, which specifies membrane localization and post-translational modifications).
The position of the RAS residues (glycine 12, glycine 13, and glutamine 61) most commonly
targeted by point mutations in thyroid cancer is indicated. BRAF: RBD (RAS-binding domain),
CRD (cysteine-rich domain), KD (kinase domain). The position of the breakpoint (break) of BRAF
in fusions identified in papillary thyroid cancer and the most common BRAF point mutation in
thyroid carcinoma (valine-to-glutamate mutation at residue 600) are indicated. Scale bars for each
protein are reported on the right end side

targeting a large number of transcription factors (Roberts and Der 2007; Schubbert
et al. 2007). Different components of the ERK system are targeted by genetic lesions
in TC (Fig. 1). As discussed in the next paragraphs, these lesions include: (i) single
base substitutions (SBS), small insertion/deletions (indels) or rearrangements of
RET or other RTKs (NTRK, ALK, and others); (ii) SBS, indels, and, more rarely,
rearrangements of BRAF; and (iii) SBS of RAS.
The PI3K pathway is another signaling circuit that is commonly upregulated in
TC, particularly in the most aggressive forms (Fig. 1) (Fagin and Wells 2016; Xing
et al. 2013). Similarly to the MAPK system, also PI3K activation is initiated by a
RTK or directly by RAS (Mendoza et al. 2011). PI3K produces the second intracel-
lular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The kinase PDK1
is activated by PIP3 binding and mediates activation of the AKT/PKB (hereafter
referred to as AKT) serine/threonine kinase via phosphorylation of its threonine 308.
The PTEN (phosphatase and tensin homolog) phosphatase functions as a gatekeeper
Pathogenesis of Thyroid Carcinoma 9

of this circuit by hydrolyzing PIP3. Active AKT leads to the inhibition of tuberous
sclerosis complex 2 (TSC2), thereby activating Rheb (Ras homolog enriched in
brain) small GTPase, which, finally, activates the serine/threonine kinase mTOR
(mammalian target of rapamycin), in the context of the mTORC1 complex. mTOR
triggers protein synthesis, cell growth, and proliferation by phosphorylating p70-S6
kinase (p70S6K or S6K1) that, in turn, phosphorylates the ribosomal protein S6 and
the eukaryotic translation initiation factor 4E–binding protein 1 (4E–BP1) (Hay and
Sonenberg 2004; Moritz et al. 2010). In a positive feedback loop, AKT is also
activated by serine 473 phosphorylation mediated by mTOR in the context of the
mTORC2 complex. Different components of the PI3K signaling system are targeted
by genetic lesions in TC (Fig. 1). As discussed in the next paragraphs, besides those
targeting RTKs and RAS, these include single base substitutions (SBS) of the
catalytic subunit of PI3K (PI3KCA) or of AKT and inactivating mutations of PTEN.
As described hereafter, genetic lesions targeting pathways other than the ERK
and PI3K ones are also found in specific subtypes of TC. These include PAX8-
PPARG fusion (mainly in FTC), THADA gene fusions (Drieschner et al. 2007), and
mutations in TP53, EIF1AX, TERT promoter, or in components of mismatch repair,
histone methyltransferase, and SWI/SNF chromatin remodeling systems (mainly in
PDTC and ATC) (Fagin and Wells 2016).

Molecular Lesions in Papillary Thyroid Carcinoma (PTC)

Papillary thyroid carcinoma (PTC) is the most common TC subtype and the
increased overall incidence of TC recorded in the past few years is mainly due to
increased detection of PTC (Cabanillas et al. 2016; Fagin and Wells 2016). Several
clinicopathological variants of PTC are identified. Small volume PTC (<1 cm) are
called microcarcinomas. Approximately 30% of the PTC cases belong to classical
(CV-PTC) variant, characterized by papillary architecture and specific nuclear fea-
tures (grooves, pseudoinclusions, and optical clearing) (Giordano 2016). Follicular
(FV-PTC) variant accounts for another 30% of the PTC cases and is characterized by
the presence of nuclear features in the context of a follicular growth pattern
(Giordano 2016; Asa et al. 2015). Rare and aggressive PTC variants include tall
cell (TCV-PTC), columnar cell, hobnail cell, and diffuse sclerosing variants (Papp
and Asa 2015).
As shown in Fig. 2, molecular pathogenesis of PTC is dominated by genetic
lesions of the ERK pathway; these lesions are alternative PTC driver events as they
generally do not overlap in individual patients (Fagin and Wells 2016). Accordingly,
the analysis of several hundred PTC samples by the Cancer Genome Atlas (TCGA)
has demonstrated that the most common PTC lesions are those targeting BRAF
(about 60% of the cases), mainly the V600E point mutation, and RAS (about 15% of
the cases), mainly point mutations in NRAS and HRAS (Fig. 3) (Cancer Genome
Atlas Research Network 2014). Most BRAF- or RAS-negative cases bear chromo-
somal rearrangements affecting RTKs (about 10% of the cases) like RET (the
receptor for glial-derived neurotrophic factor family of neurotrophic growth factors:
10 M. Santoro and F. Carlomagno

GDNF, NRT, ART, PSP) or, less frequently, ALK, NTRK1, NTRK3, or others
(Fagin and Wells 2016; Cancer Genome Atlas Research Network 2014). These
rearrangements typically cause the fusion of the 50 -terminal portion, containing an
active transcriptional promoter and a protein dimerization encoding motif, of one of
several different genes, to the 30 -portion of a RTK gene encoding the catalytic
domain. In the case of RET, these rearrangements have been termed “RET/PTCs,”
the most common variants being represented by RET/PTC1 (the CCDC6-RET
fusion) and RET/PTC3 (the NCOA4-RET fusion) (Fig. 3) (Santoro and Carlomagno
2013).
Ionizing radiation-associated childhood PTC cases, in particular, those occurred
early after the Chernobyl disaster, have a very high prevalence (up to 80%) of fusion
oncogenes involving RET or other (other RTKs, BRAF or PPARG) genes (Ricarte-
Filho et al. 2013). Accordingly, exposure to X-rays induces RET/PTC fusions in
cultured thyrocytes (Ito et al. 1993).
The TCGA study has subdivided PTC in two major classes named BRAFV600E-
like (BVL) and RAS-like (RL) PTC. BVL-PTC include samples with BRAFV600E
mutation (or more rarely BRAF fusions), low differentiation, with high levels of
MAPK signaling, and enriched for classical (CV-PTC) and tall cell (TCV-PTC)
subtypes (Fagin and Wells 2016; Cancer Genome Atlas Research Network 2014;
Ciampi et al. 2005). RL-PTC include samples with RAS (NRAS>HRAS>KRAS)
mutations, well differentiated, and belonging to the follicular variant (FV-PTC)
(Fagin and Wells 2016; Cancer Genome Atlas Research Network 2014; Zhu et al.
2003). When positive for BRAF lesions, FV-PTC commonly harbor the K601E
mutation or mutations other than the V600E one (Afkhami et al. 2016). Mechanis-
tically, the intensity of MAPK signaling (higher in the case of BRAF V600E and
BRAF fusions and lower in the case of other BRAF lesions or RAS mutations)
correlates with the different tumor phenotypes (Cancer Genome Atlas Research
Network 2014). Similar to FTC, FV-PTC may also harbor the PAX8-PPARG fusion
(1–5% of the cases) (Armstrong et al. 2014).
Thus, the morphological (follicular architecture) and genetic similarities render
FV-PTC closely related to the FTC category (Giordano 2016; Asa et al. 2015).
FV-PTC can be infiltrative or encapsulated (Hodak et al. 2016). Encapsulated
FV-PTC have an indolent disease course, and it has been recently proposed that
these tumors should be renamed NIFTP (noninvasive follicular thyroid neoplasms
with papillary-like nuclear features) and handled as neoplasms with very low
malignant potential (Hodak et al. 2016; Nikiforov et al. 2016).

Molecular Lesions in Follicular Thyroid Carcinoma (FTC)

Tumors that recapitulate the follicular architecture of the normal gland, and that lack
the nuclear features of PTC, are divided into follicular thyroid adenomas (FTA),
which are noninvasive, and follicular thyroid carcinomas (FTC), that demonstrate
capsular and/or vascular invasion (Giordano 2016). Similar to FV-PTC, FTC are
characterized by mutually exclusive SBS in RAS (approximately 50% of the cases),
Pathogenesis of Thyroid Carcinoma 11

the PAX8-PPARG (peroxisome-proliferator activated receptor gamma), or other rare

PPARG variant fusions (approximately 35% of the cases) (Fagin and Wells 2016;
Nikiforova et al. 2003). The PAX8-PPARG fusion (also referred to as PPFP) arises
as the consequence of a reciprocal translocation between chromosome regions 2q13
and 3p25. This juxtaposes the PAX8 transcriptional promoter (that is highly active in
thyrocytes) and the PAX8 coding sequence (just missing the C-ter activating
domain) to the entire PPARG coding sequence (Eberhardt et al. 2010; Raman and
Koenig 2014). PAX8 is a transcription factor of the paired box family that is
necessary for thyroid development (Pasca di Magliano et al. 2000). PPARG is a
member of the nuclear receptor family of transcription factors and it is involved in
regulation of adipogenesis and insulin sensitivity (Krishnan et al. 2007). The PPFP
chimeric protein exerts oncogenic activity, probably as the consequence of its
dominant negative inhibitor activity on endogenous PPARG and perturbed regula-
tion of PAX8- and PPARG-regulated genes (Raman and Koenig 2014). FTC positive
for PAX8-PPARG fusion present at a younger age are smaller and almost always
overtly invasive with respect to RAS mutant ones (Nikiforova et al. 2003).
It is still unknown whether FTA has any potential of evolving into malignant
lesions (Raman and Koenig 2014). Approximately half of them have RAS muta-
tions; less than 5% have the PAX8-PPARG fusion (Nikiforova et al. 2003). Among
the three RAS oncogenes (KRAS, HRAS, NRAS), the KRAS codon 12/codon13
mutation was found associated with a significantly lower carcinoma risk than HRAS
codon 61 and NRAS codon 61 mutations (Radkay et al. 2014).
Hürthle cell (oncocytic) carcinomas (HCC) are considered FTC variants; how-
ever, only infrequently they feature FTC-related genetic lesions, e.g., RAS mutations
or PAX8-PPARG fusion (Nikiforova et al. 2003). Mutations of GRIM-19 gene, a
component of complex I mitochondrial chain, as well as of other proteins of this
complex encoded by the mitochondrial DNA, have been described in HCC
(Nikiforov and Nikiforova 2011; Máximo et al. 2005; Gasparre et al. 2007). A
particular type of genomic instability has been found in HCC, with copy number
gains of large regions of chromosomes 5, 7, 12, and 17 that can represent the genetic
driver lesion for this type of tumor (Ganly et al. 2013). Moreover, gene expression
profiling has highlighted a potential role of the PI3K pathway in HCC (Ganly et al.
2013).

Molecular Lesions in Anaplastic (ATC) and Poorly Differentiated

(PDTC) Thyroid Carcinoma

Though rare, poorly differentiated, and anaplastic thyroid carcinomas represent an

important clinical issue, being intrinsically refractory to radiometabolic treatment
(Smallridge et al. 2009; Smallridge and Copland 2010). Criteria for PDTC diagnosis
listed in the Turin proposal include architectural and high-grade (mitosis and necro-
sis) features, solid/nested/insular pattern of growth, absence of nuclear features of
PTC, and presence either of convoluted nuclei, or mitotic activity 3  10 high
power fields, or tumor necrosis (Volante et al. 2007). PDTC and ATC frequently
12 M. Santoro and F. Carlomagno

Table 1 Phenotype of MEN2 syndromes

Other
Syndrome Neoplasms manifestations
MEN2A Medullary Pheochromocytoma Parathyroid Hirschsprung’s
thyroid hyperplasia disease
carcinoma Cutaneous
lichen
amyloidosis
MEN2B Medullary Pheochromocytoma Ganglioneuromatosis Marfanoid
thyroid habitus
carcinoma Ocular
abnormalities

arise from pre-existing DTC, as indicated by the patients’ history, presence of areas
of differentiation within the cancer bulk, and the overlapping of some molecular
lesions with those present in DTC. Accordingly, BRAFV600E has been found in
33% and 45% of PDTC and ATC, respectively, while RAS gene mutations have
been found in approximately 25% of both PDTC and ATC (Landa et al. 2016;
Kunstman et al. 2015) (Fig. 2). RAS mutations were overrepresented in PDTC
fulfilling the Turin criteria, while BRAFV600E was overrepresented in PDTC only
featuring high mitotic rate and necrosis (irrespective of the growth pattern) (Landa
et al. 2016).
As shown in Fig. 2, progression from DTC is believed to depend on the
acquisition of additional mutations. Accordingly, mutations exclusively associated
with PDTC and ATC, and thus virtually absent in DTC, are known. These include
TP53 mutations in up to 70% ATC (Landa et al. 2016; Fagin et al. 1993), mutations
in PI3K pathway components (Ricarte-Filho et al. 2009), mutations in the WNT
signaling pathway (CTNNB1/βCatenin, AXIN1 and APC) (Garcia-Rostan et al.
1999), and mutations in the TERT transcriptional promoter (Landa et al. 2016;
Landa et al. 2013).
TERT (telomerase-reverse transcriptase) represents the catalytic subunit of the
telomerase complex whose activity is necessary to overcome telomeric DNA short-
ening and replicative senescence (Kumar et al. 2016). Mutations in TERT transcrip-
tional promoter have been found in melanoma, urothelial bladder and liver cancer,
and glioblastoma (Kumar et al. 2016; Vinagre et al. 2013). Most common mutations
are the cytidine-to-thymidine transitions named C228T and C250T. These mutations
have a gain-of-function effect releasing TERT from the epigenetic silencing that
normally occurs in adult cells by promoting recruitment to the TERT promoter of the
GA-binding protein (GABP) transcription factor; GABP, in turn, belongs to the ETS
(E-twenty six) family transcription factors that are prototypic MAPK-responsive
factors (Akıncılar et al. 2016; Liu et al. 2016a). Importantly, TERT mutations
co-occurred with BRAF and RAS mutations in PDTC and ATC (Landa et al.
2016; Melo et al. 2014). Importantly, co-occurrence of BRAF and TERT mutations
correlated with increased mortality in PTC patients (Liu et al. 2016b).
Pathogenesis of Thyroid Carcinoma 13

Recent genome-wide next-generation sequencing approaches have contributed to

the identification of further genetic lesions and novel pathways involved in PDTC
and ATC (Landa et al. 2016; Kunstman et al. 2015) (Fig. 2). Alterations of additional
players in the ERK pathway, such as mutations of NF1 and deletions of NF2, have
been identified (Landa et al. 2016). NF1 (Neurofibromatosis 1) codes for a tumor
suppressor called Neurofibromin, a GTPase-activating protein (GAP) that functions
as a gatekeeper of RAS activity (Schubbert et al. 2007). NF2 (Neurofibromatosis 2)
codes for a tumor suppressor called Merlin that negatively regulates the Hippo-YAP
signal transduction pathway. Thus, Merlin loss in TC unleashes the ability of the
YAP transcription factor to stimulate RAS genes expression (Garcia-Rendueles et al.
2015). The EIF1AX gene codes for a eukaryotic translation initiation factor that is
required for the transfer of methionyl-tRNA molecules to ribosomes in order to
initiate protein translation. Mutations in EIF1AX have reported in about 1% of PTC
in a mutually exclusive manner with respect to BRAF and RAS mutations (Cancer
Genome Atlas Research Network 2014). EIF1AX mutations are more common in
PDTC and ATC (approximately 10% of the cases) and are strongly associated with
RAS mutations (Landa et al. 2016; Kunstman et al. 2015).
Besides ERK and PI3K signaling networks, novel oncogenic pathways have been
identified in PDTC and ATC (Fig. 2). Besides TP53 mutations, alterations of several
other genes encoding proteins involved in DDR (DNA damage response) are present
in advanced TC, probably concurring to its genomic instability. They include players
of the mismatch repair (MMR) pathway, such as MSH2, MSH6, MLH1, and MLH3,
and, more rarely, the checkpoint kinase ATM, involved in DNA double-strand break
repair. In addition, genes encoding proteins involved in the epistatic control of gene
expression and chromatin remodeling are frequently mutated in ATC. As an exam-
ple, components of the SWI/SNF complex (ARID1A, ARID2, ARID5B,
SMARCB1, PBMR1, and ATRX) are mutated in 36% of ATC and 6% of PDTC,
and diverse histone methyltransferases (KMT2A, KMT2C, KMT2D, and SETD2)
are mutated in 24% of ATC and 7% of PDTC. The acetyltransferase CREBBP has
been frequently found altered in advanced thyroid cancer (Landa et al. 2016;
Kunstman et al. 2015). Finally, somatic gene copy number alterations are much
more represented in advanced thyroid cancer, particularly ATC (mainly 8p and 17p
losses and 20q gains) than in DTC (Kunstman et al. 2015).
As previously discussed, gene rearrangements are found in DTC, including RET
and, less frequently, other RTK gene fusions in PTC and PAX8-PPARG fusion in
FTC. These rearrangements are scarcely represented in ATC indicating that, for as
yet unknown reasons, they are associated to cancers that have a reduced propensity
to progression (Kunstman et al. 2015).

Molecular Lesions in Medullary Thyroid Carcinoma (MTC)

Medullary thyroid carcinoma (MTC) is a rare cancer that originates from thyroid
parafollicular C cells that are responsible for the synthesis of the calcitonin (Wells
et al. 2013; Tuttle et al. 2014). Recent studies have attributed to anterior endoderm
14 M. Santoro and F. Carlomagno

rather than neuroectoderm the origin of C cells (Johansson et al. 2015). MTC can be
either sporadic (75% of cases) or familial (25% of cases). Familial cases occur as part
of the autosomal dominant inherited cancer syndrome identified as multiple endo-
crine neoplasia type 2 (MEN2) (Wells et al. 2013; Tuttle et al. 2014). In MEN2,
MTC can be isolated or associated to other phenotypes that delineate two different
MEN2 subtypes: MEN2A (the most common one) and MEN2B (Table 1). Besides
MTC, MEN2A patients can also display pheochromocytoma and parathyroid hyper-
plasia; more rarely, they develop cutaneous lichen amyloidosis (CLA),
Hirschsprung’s disease (HD), or prominent corneal nerves (Wells et al. 2013).
Besides MTC, MEN2B patients develop pheochromocytoma, generalized
ganglioneuromatosis, marfanoid habitus, and ocular abnormalities (Wells et al.
2013). FMTC (familial MTC) is characterized by the presence of only MTC;
recently, FMTC has been proposed to be a variant of MEN2A (Wells et al. 2015).
In MEN2 patients, MTC is preceded by a preneoplastic C cell hyperplasia which is
generally present in the first decade in MEN2A patients or at birth in MEN2B ones
and can be revealed by increased levels of pentagastrin-stimulated serum calcitonin
(Machens et al. 2003). Serum calcitonin levels are also used to monitor persistent or
recurrent disease and response to therapy, as they are directly proportional to tumor
bulk. CEA (carcinoembryonic antigen) is another tumor marker for MTC (Wells
et al. 2013).
The gene responsible for MEN2 maps on chromosome 10 (10q11.2) and encodes
the receptor tyrosine kinase RET (Santoro and Carlomagno 2013). More than
100 different germline point mutations of RET gene have been found in MEN2
patients, accounting for over 95% of carriers. In most MEN2A cases, specific
extracellular cysteines in the cysteine-rich domain of RET (C609, 611, 618, 620 in
exon 10 and C634 in exon 11) (Fig. 3) are mutated to different amino acids, with
C634 mutations mainly associated to the MEN2A complete phenotype (MTC,
pheochromocytoma, and parathyroid hyperplasia). Mutations of other residues,
mainly in exons 13, 14, and 15 of RET, can also be associated to MEN2A and
FMTC (Fagin and Wells 2016; Wells et al. 2013). MEN2B is caused by mutations in
RET exon 16 (M918 T) (Fig. 3), less often exon 15 (A883F), or, rarely, double RET
mutations (e.g., V804M plus Y806C or, alternatively, E805K, S904C, Q781R)
(Fagin and Wells 2016; Wells et al. 2013; Frank-Raue et al. 2011). Genetic testing
in MEN2 family members is mandatory to identify carriers that can be treated with
preventive thyroidectomy (Wells et al. 2013). Such screening is important also in
sporadic cases because some of them are, in fact, familial (Elisei et al. 2007). RET
MEN2 mutations convert RET in a dominant oncogene by activating RET auto-
phosphorylation and downstream signaling in a ligand-independent manner and
generating, indeed, a compelling therapeutic target for MEN2-associated tumors
(Santoro and Carlomagno 2013; Mulligan 2014).
Approximately half of sporadic MTC also carry RET mutations, prevalently
M918T, and more rarely other RET SBSs (like V804M/L) or Indels or finally, as
recently described, RET fusion to the MYH13 gene (Fagin and Wells 2016; Wells
et al. 2013; Grubbs et al. 2015). RET-negative sporadic MTC bear commonly
activating mutations in RAS (principally HRAS and KRAS) genes (Moura et al.
Pathogenesis of Thyroid Carcinoma 15

2011; Boichard et al. 2012; Ciampi et al. 2013; Agrawal et al. 2013; Ji et al. 2015).
Additional rare driver events, still targeting the ERK cascade, have been identified in
RET- and RAS-negative MTC cases, such as fusions of GFPT1 and EML4 genes to
the ALK RTK (Ji et al. 2015) and fusion of PARP12 to the BRAF gene (Kasaian
et al. 2016). Besides those described above, other recurrent driver mutations have not
yet been described in MTC (Agrawal et al. 2013; Ji et al. 2015).

Conclusions

Recent studies, mostly based on next-generation sequencing approaches, have

contributed to depict the genetic landscape of the major thyroid cancer histotypes
such as PTC, FTC, PDTC, ATC, and MTC (Fagin and Wells 2016). Besides the ERK
and PI3K pathways, novel genetic players have emerged in thyroid tumorigenesis
which includes proteins involved in translation, mismatch repair, and in chromatin
remodeling (Cancer Genome Atlas Research Network 2014; Landa et al. 2016;
Kunstman et al. 2015).
Translation of this knowledge to clinical practice is being very rapid. Probably,
the most striking example of this concept is the diagnosis of MEN2 carriers based on
the identification of germline RET mutations (Wells et al. 2015). In addition, a
negative prognostic role of the BRAF V600E mutation which is associated to
increased risk of nodal recurrences for PTC has been proposed by some authors
(Xing et al. 2015). Novel molecular tests either based on a gene expression classifier
or next-generation sequencing of a panel of TC driver genes have been proposed to
be able to complement pathology diagnosis of TC (Hsiao and Nikiforov 2014).
Finally, molecular characterization of TC has encouraged the initiation of several
targeted therapy clinical trials which have led to the approval of novel drugs for
radioiodide refractory TC (lenvatinib and sorafenib) or for locally advanced or
metastatic MTC (vandetanib and cabozantinib) (Fagin and Wells 2016; Haraldsdottir
and Shah 2014; Dunn and Fagin 2015). It is feasible that these progresses may
continue to be translated into better diagnosis and treatment of TC in the next future.

Summary

Thyroid cancer is classified in different subtypes based on the type of cell of origin
(C cell or follicular cell) and on the expression of differentiation markers. Several
genetic lesions have been associated to the different types of thyroid cancer, with the
RAS/MAPK pathway almost invariably activated in all types. Advanced thyroid
cancers display additional mutations in pathways controlling genome stability,
survival and senescence. Such distinctive features might help designing new thera-
pies and diagnostic strategies.
16 M. Santoro and F. Carlomagno

Cross-References

▶ Thyroid Nodule
▶ Differentiated Thyroid Carcinoma of Follicular Origin
▶ Medullary Carcinoma
▶ Anaplastic and Other Forms of Thyroid Carcinoma
▶ New (Medical) Treatment for Thyroid Carcinoma

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