REVIEW Open Access [606500]

REVIEW Open Access
New insights into the diagnosis of nodular goiter
Anhelli Syrenicz1*, Monika Kozio łek1, Andrzej Ciechanowicz2, Anna Sieradzka1, Agnieszka Bi ńczak-Kuleta2
and Mi łosz Parczewski3
Abstract
Preoperative diagnostic investigations of nodular goiter are based on two main examinations: ultrasonography of
the thyroid gland and ultrasound-guided fine-needle aspiration biopsy. So far, FNAB has been the best method for
the differentiation of nodules, but in some cases it fails to produce a conclusive diagnosis. Some of the biopsies do
not provide enough material to establish the diagnosis, in some other biopsies cytological picture is inconclusive.
Determining the eligibility of thyroid focal lesions for surgery has been more and more often done with molecular
methods. The most common genetic changes leading to the development of thyroid cancer include mutations,
translocations and amplifications of genes, disturbances in gene methylation and dysregulation of microRNA. The
mutations of Rasproto-oncogenes and BRAF gene as well as disturbances of DNA methylation in promoter regions
of genes regulating cell cycle (e.g. hypermethylation of RASSF1A gene and TIMP-3 gene) play an important role in
the process of neoplastic transformation of thyreocyte. The advances in molecular biology made it possible to
investigate these genetic disturbances in DNA and/or RNA from peripheral blood, postoperative thyroid tissue
material and cytology specimens obtained through fine-needle aspiration biopsy of focal lesions in the thyroid gland.
As it became possible to analyze the mutations and methylation of genes from cell material obtained through
fine-needle aspiration biopsy, it would be beneficial to introduce the techniques of molecular biology in the
pre-operative diagnosis of nodular goiter as a valuable method, complementary to ultrasonography and FNAB.
The knowledge obtained from molecular studies might help to determine the frequency of follow-up investigations in
patients with nodular goiter and to select patients potentially at risk of developing thyroid cancer, which would
facilitate their qualification for earlier strumectomy.
Keywords: Nodular goiter, Thyroid cancer, Genetic testing
Introduction
Nodular goiter is the most common pathology of the
thyroid gland. Palpable thyroid nodules are found in
3-7% of adult population and are more frequent in
women. Ultrasonography which has been introduced in
the diagnosis of thyroid gland has confirmed earlier aut-
opsy reports indicating that focal lesions are found in as
many as 50% of clinically normal thyroid glands [1,2].
Preoperative diagnostic investigations of nodular goiter
are currently based on two main examinations: ultraso-
nography of the thyroid gland and ultrasound-guided
fine-needle aspiration biopsy. Determining the eligibility
of thyroid focal lesions for surgery has been more and
more often done with molecular methods providing theinformation on possible presence of mutations and epi-
genetic changes which play an important role in malig-
nant transformation [3-12]. Most focal lesions in the
thyroid gland are of benign nature. The incidence of thy-
roid cancer in multinodular goiter is estimated at ap-
prox. 5-10%. Thyroid cancer is more common in solitary
thyroid nodules (approx. 10-20%) than in multinodular
goiter [1,13]. Clinical examination of patients with thy-
roid nodules remains important component of cancer
risk assessment. The risk factors include positive family
history (this is especially true for medullary carcinoma
and some papillary carcinomas), age (under 20 years and
over 60 years), sex (males are at greater risk) and history
of head and neck irradiation, particularly in the child-
hood. Other very important symptoms include dyspho-
nia, raucity and neck pain in patients with hard, not
easily movable lump [14,15].* Correspondence: klinendo@sci.pum.edu.pl
1Department of Endocrinology, Metabolic Diseases and Internal Diseases,
Pomeranian Medical University in Szczecin, Szczecin, Poland
Full list of author information is available at the end of the article
© 2014 Syrenicz et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.Syrenicz et al. Thyroid Research 2014, 7:6
http://www.thyroidresearchjournal.com/content/7/1/6

Ultrasonographic examination of focal thyroid lesions,
particularly those in multinodular goiter is helpful in
selecting one or more foci for fine-needle aspiration bi-opsy. It is currently believed that the most important cri-
teria for the evaluation of malignant potential of a thyroid
nodule is not its size but rather its vascularization, thepresence of microcalcifications, height/width ratio, struc-
ture (solid or solid-fluid), echogenicity and border margins
as well as the presence of so called halo. Hence, thyroidnodules most suspected of malignancy are those with the
following ultrasonographic features: enhanced central vas-
cularization or no flow in power doppler; microcalcifica-tions; nodule height exceeding its width; solid lesions are
more suspicious than solid-fluid ones; hypoechogenic le-
sions raise more concerns than isoechogenic ones; lesionswith rough margins and those without a halo or lesions
with irregular, thick halo are more suspicious [16]. Now-
adays, ultrasound-guided fine-needle aspiration biopsy is agold standard in the diagnosis of nodular goiter. It is tech-
nically simple, safe and inexpensive. Cytology assessment
of the specimens obtained through fine-needle aspirationbiopsy is based on international classification known as
the Bethesda System of Reporting Thyroid Cytopathology
[17]. According to this classification, the findings of fine-needle aspiration biopsy of thyroid nodule can be divided
into 6 groups of diagnostic cytopathology categories: I-
non-diagnostic or unsatisfactory, II- benign, III- atypia ofundetermined significance or follicular lesion of undeter-
mined significance, IV- follicular neoplasm or suspicious
for a follicular neoplasm, V- suspicious for malignancyand VI- malignant. Cytopathology diagnoses falling within
groups IV, V and VI are indications for surgery. Diagnoses
classified as group III and those classified as group I meanthat fine-needle aspiration biopsy should be repeated. It is
also worth emphasis that even the diagnosis of a benign
lesion (group II ) in fine-needle aspiration biopsy carries3% risk of false negative result [17]. Irrespective of the
chosen classification of cytopathology findings, one should
assume that approx. 20% of thyroid nodule biopsies pro-duce results that require final diagnosis based on postop-
erative histopathology report [13].
The scintigraphy of the thyroid gland is currently con-
sidered less useful in the diagnosis of possible malignancy
in the nodular goiter since it has been demonstrated thatmalignant lesions may be found not only in cold nodules
but also in approx. 3% of solitary hot thyroid nodules [18].
Thyroid cancer is the most common malignancy of
endocrine system accounting for 2.5% of all cancers di-
agnosed in humans [19]. It has been diagnosed more
frequently since 1990s, particularly in women [20]. InPoland, there are 1500 to 1800 new cases of thyroid can-
cer per year [21]. The most common histological type of
thyroid cancer is papillary thyroid carcinoma (PTC) andthe second most common differentiated thyroid canceris follicular thyroid carci noma (FTC); they account for
80% and 15% of all thyroid cancers, respectively [22,23].
PTC and FTC are both classified as differentiated can-cers of the thyroid gland, deriving from highly diffe-
rentiated follicular cell which is dedifferentiated in the
presence of various factors, fails to undergo apoptosisand becomes capable of uncontrolled proliferation and
f o r m i n gm e t a s t a s e s[ 2 4 , 2 5 ] .N e o g e n e s i si sam u l t i s t a g e
process, involving multiple genes as well as endo- andexogenous factors. At the ini tial stage of neogenesis
called initiation, the cell is damaged. The second stage
called promotion is the clonal expansion of abnormalcell triggered by mitogenic signal, which produces a
group of dividing cells with still benign genotype. The
third stage is neoplastic transformation of some clonalcells caused by further genetic changes –oncogenesis is
part of progression stage.
Review
The most common genetic changes resulting in the deve-
lopment of thyroid cancer are mutations, translocationsand amplifications of genes, disturbances in gene methy-
lation and dysregulation of microRNA [3,4,6,26]. The ad-
vances in molecular biology made it possible to investigatethese genetic disturbances in DNA and/or RNA from per-
ipheral blood, postoperative thyroid tissue material and
cytology specimens obtained through fine-needle aspir-ation biopsy of focal lesions of the thyroid gland. They cast
new light on the genesis of benign and malignant lesions
in the thyroid gland and also opened new perspectives forpreoperative diagnosis of focal thyroid lesions. The first
Polish center to conduct molecular tests of cytology ma-
terial obtained from fine-needle aspiration biopsy of thethyroid gland was in Szczecin and these tests were aimed
at the detection of somatic mutations of TSH receptor
gene and G protein alpha chain [27,28]. New moleculardiagnostic tools applied to fine-needle aspiration biopsy
allowed for more precise qualification of patients for total
or partial strumectomy. The key role in the neoplastictransformation of thyroid follicular cell is played by
the inactivation of suppression genes and activation of
oncogenes [3,4,24,25,29]. Mutations observed in thyroidcancers usually affect RAS, BRAF, PTEN, CTNNB1, TP53,
IDH1, ALK and EGFR genes [9,12,25,30].
BRAF gene plays a very important role in the etio-
pathogenesis of papillary thyroid carcinoma [7,8,31-33].
The predominant BRAF gene mutation reported in PTC,
observed in 36-80% of PTC cases is the thymine-to-adenine transversion at position 1799 (T1799A) in exon
15, resulting in the substitution of valine (V) by glutamic
acid (E) at codon 600 (V600E) [22,33-40]. This specificV600E BRAF mutation represents 99% of all BRAF muta-
tions found in thyroid cancer [6]. Many studies prove that
this mutation is found only in papillary thyroid carcinomaSyrenicz et al. Thyroid Research 2014, 7:6 Page 2 of 7
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and in few cases of anaplastic cancer [34,36,41]. The pre-
sence of BRAF T1799A oncogene is an unfavorable
prognostic factor in PTC as it increases the aggressivenature of cancer through raising its invasiveness, ac-
celerating relapses and the occurrence of metastases
[23,33,34,36,37,42-44] BRAF gene encodes BRAF pro-
tein. BRAF protein belongs to a class of serine/threonine
kinases and subfamily of RAF proteins [8,45,46]. Cytoplas-
mic RAF proteins make up RAS-RAF-MEK-ERK pathwaywhich is involved in the transduction of mitogenic signal
from the cell surface to cell nucleus [47-49]. This pathway
using tyrosine kinase receptor is a mitogen-activated ki-nase cascade called MAPK (Mitogen-Activated Protein
Kinase) [32,47-49]. BRAF gene mutation activating MAPK
pathway is most likely the main contributor to the devel-opment and progression of PTC. T1799A BRAF oncogene
is present at all stages of PTC progression, it may even be
there at early stages of the development of micropapillarycancer [6,31,34,50]. The presence of BRAF mutation in
cytology material obtained from FNAB of thyroid nodule
indicates the necessity of surgical treatment. It should beremembered, however, that false positive results indicating
the presence of BRAF in the nodule are reported in
0.2-5.7% of cases, while false negative results are found in1.9-5.8% of cases [51-54]. Since in some cases of thyroid
nodule biopsy, the specimens for cytology assessment do
not contain enough cells with mutated BRAF and theresult obtained is negative despite the mutation in
thyroid nodule, currently there are indications to re-
evaluate BRAF mutation in follow-up biopsy performed afew months later, especially when the nodule presents
ultrasonography features of malignancy and cytology as-
sessment of focal thyroid lesion gives non-diagnostic re-sults, there are signs of atypia or the nodule is of benign
nature [11].
Following BRAF mutation, the second most common
mutation observed in thyroid cancer are the mutations
of RAS proto-oncogenes, which play an important role
in the initiation of thyreocyte neoplastic transform-ation [12,55,56]. Proto-oncogenes of RAS family (N-RAS,
H-RAS, K-RAS) are located on 1, 11 and 12 chromosome,
respectively. They are involved in the control of growth
and differentiation of cells. They encode G membrane
proteins showing intrinsic GTP-ase activity and parti-cipate in signal transmission from membrane tyrosine
kinase receptor to cell nucleus using both MAPK and
PI3K-AKT pathways. Mutat ions at codons 12, 13 and
61 transform these proto-oncogenes into active onco-
genes [6,57]. Mutations of RAS proto-oncogene make
the protein encoded by this proto-oncogene lose its intrin-sic GTP-ase activity and there is a constitutive activation
of signal transduction pathway. In thyroid tumorigenesis,
PI3K-AKT is a preferable pathway [55]. RAS gene muta-tions are observed both in benign and malignant thyroidneoplasms. These mutations can be found in 40-50% of
FTC cases, in 5-20% of PTC cases, in 20-40% of poorly
differentiated and anaplastic cases as well as in ap-prox. 30% of follicular adenomas [9,32,35,56,58,59]. RAS-
positive follicular adenoma may be a precursor of both
follicular cancer and follicular variant of papillary carcin-oma [6]. Recent studies have emphasized an important
role of RAS mutation as a valuable diagnostic marker in
tumors with very difficult or impossible diagnosis basedon cytology assessment of fine-needle aspiration biopsy
material, which is true for follicular variant of papillary
cancer and follicular adenoma [60,61]. Diagnostic difficul-ties with follicular variant of papillary cancer result from
the absence of papillary proliferation and limited nuclear
features typical for papillary cancer. To differentiate be-tween adenoma and follicular cancer, the assessment of
vascular invasion and capsule infiltration is necessary,
which cannot be based on cytology material obtained fromfine-needle aspiration biopsy [39,56]. Most RAS-positive
thyroid nodules with indeterminate cytology and without
suspicious ultrasonography features turn out to be afollicular form of papillary cancer with low level of ma-
lignancy in post-operative histopathology examination
[12]. RAS mutations are found in a large percentage ofpoorly differentiated and anaplastic thyroid cancers, there-
fore, it seems advisable to co nsider surgical treatment
of all RAS-positive thyroid nodules to prevent cancerprogression [12].
Yet another kind of genetic changes found in thyroid
cancer are oncogenic rearrangements resulting from genetranslocations, with RET/PTC and PAX8/PPAR γbeing the
most common [62-64]. More than 10 types of RET/PTC
translocation have been described but the two most fre-quently occurring are RET/PTC1 and RET/PTC3 [62,65].
RET is a proto-oncogene encoding RTK. RET/PTC is
formed as a consequence of genetic recombination be-tween the 3 ’portion of RET tyrosine kinase and the 5 ’
portion of a partner gene. In the case of RET/PTC1 re-
arrangement, the partner gene is CCDC6 known as H4(coiled-coil domain-containing gene 6), while in RET/
PTC3 rearrangement, the partner gene is NCOA4 known
as ELE1 (nuclear receptor co-activator 4). The structural
basis for RET/PTC transformation is close vicinity of RET
and the partner gene in cell nucleus [66,67]. The conse-quence of this rearrangement is ligand-independent dime-
rization and constitutive activation of RET tyrosine kinase
[25]. RET/PTC1 is the most frequent type accounting for60-70% of re-arrangements and RET/PTC3 is observed in
20-30% of PTC cases [68]. RET/PTC1 re-arrangements
are more common in classic forms of papillary cancer andin papillary microcarcinoma [3,69]. On the other hand,
RET/PTC3 re-arrangements are more common in solid
papillary cancers, which was observed especially in theUkraine and Belarus after Chernobyl disaster [70].Syrenicz et al. Thyroid Research 2014, 7:6 Page 3 of 7
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Another important re-arrangement observed in thy-
roid cancer is PAX8/PPAR γas a consequence of the
translocation of genetic material between chromosomes2 and 3. Then, PAX8 gene which encodes thyroid-specific
transcription factor domain is combined with PPAR γ(per-
oxisome proliferator-activated receptor- γ). PAX8/PPAR γ
rearrangement is observed mainly in follicular cancers,
but also in the follicular form of papillary cancer and fol-
licular adenomas [64,71].
It is not only the translocation of genetic material but
also the amplification of oncogenes that may play a very
important role in the thyroid tumorigenesis. This is es-pecially the case in the genes encoding MAPK path-
way kinases using tyrosine kinase receptor but also in
the genes encoding PI3K-AKT pathway. Elevated num-ber of oncogene copies are more common in anaplas-
tic cancer than in differentiated cancers of the thyroid
gland, which suggests that it may be of considerablerelevance for cancer aggressiveness and the rate of its
progression [72].
It has been demonstrated that neoplastic transform-
ation of the thyroid gland is also affected by epigenetic
mechanisms, i.e. the mechanisms influencing the regula-
tion and modification of genetic material, not affectingthe nucleotide sequence [4,5,25,29,73-75]. These mecha-
nisms include DNA methylation and histone modifi-
cation. DNA methylation takes place through covalentmodification of cytosines and it is catalyzed by DNA
methyltransferases, which attach methyl group at the
carbon 5 ’position of cytosine ring. This modification ap-
plies only to cytosines (C) which are followed by guanine
(G) in the sequence. CG sequences are grouped in the
genome sites known as CpG islands, where CG dinu-cleotide repetitions extend over 1,000 – 2,000 base pairs
[76]. In the case of genes with vital significance for fun-
damental cellular processes, with widespread expressionin the tissues, CpG islands associated with them are al-
most always found on 5 ’side of encoding sequences,
typically in the promoters of these genes. The generalmechanism of silencing expression of hypermethylation-
dependent DNA genes has several aspects. The most im-
portant of them is to prevent the binding of transcrip-
tion factors to promoters and sequences regulating
transcription, on the basis of spatial conflict. These dataare consistent with observations which imply that CpG
islands of suppressor genes in healthy somatic cells are
usually characterized by low levels of methylation or nomethylation at all. During oncogenesis, hypermethyla-
tion of these sites often occurs, which causes silencing of
their expression. Thus, in the selective strategy of a neo-plasm, hypermethylation of genes is aimed at marking
those genome areas which are to undergo deletion pro-
cesses, leading to irreversible loss of growth control. Theresult of this situation is not only accelerated growth ofcells but also the beginning of particularly dangerous
genetic instability [77,78]. The genes controlling the pro-
liferation of cells, which undergo hypermethylation inthe papillary carcinoma include TIMP-3 (tissue inhibitor
of metalloproteinase-3, inhibitor of extracellular metallo-
proteinases), DAPK (calcium-dependent protein kinase),taking part in programmed cell death, SLC5A8 (sodium
symporter), DNA repair genes (hMLH1, PCNA) and
thyroid-specific genes (NIS-sodium-iodine symporter,TSHR- thyroid stimulating hormone receptor). The genes
encoding the suppressors of neoplasia undergoing hy-
permethylation in follicular cancer include PTEN (phos-phatase inhibiting one of mitogen signal transduction
pathway), RASSF1A (signal protein of mitogen RAS path-
way), thyroid-specific genes (NIS, TSHR) and TRbeta (re-ceptor beta for thyroid hormones) [1,4,5,75,79,80]. Special
attention should be paid to TIMP-3 and RASSF1A genes
taking part in the tumorigenesis of the thyroid gland[4,5,29]. TIMP-3 inhibits the growth, angiogenesis and in-
vasion of many cancers. Hypermethylation of this gene is
particularly important in the onset of papillary thyroidcancer. It has been demonstrated that there is a correl-
ation between loss of TIMP-3 gene function as metal-
loproteinase inhibitor associated with hypermethylationand extrathyroid invasion of papillary carcinoma, lymph
node metastases and multifocal nature of this cancer
[4,75,81,82]. Protein products of RASSF1A suppressorgene participate in controlling cell cycle, controlling the
differentiation and proliferation of cells through direct
regulation of transcription and regulation of proapopticsignal pathways. Epigenetic silencing of this gene expres-
sion through promoter hypermethylation may lead to
unauthorized divisions of mutated cells. Decreased ex-pression of RASSF1A gene and/or reduced activity of its
protein products are also affected by changes in DNA se-
quence related to the acquisition of genome instability byneoplastic cells, resulting from loss of heterozygosity as
well as from the instability of microsatellite sequences
[1,4,5,29]. Hypermethylation of RASSF1A gene is ob-served both in benign thyroid neoplasms and in thy-
roid cancers, particularly in FTC. Methylation levels
above 50% of alleles was only observed in follicular thyroid
cancer, while it was not observed in benign neoplasms of
the thyroid gland, suggesting that methylation through si-lencing both RASSF1A gene alleles may play an important
role in the pathogenesis and development of follicular thy-
roid cancer [1,4,5,29].
Conclusion
New opportunities for the analysis of mutation and me-thylation of genes obtained from fine-needle aspiration
biopsy presented in this article confirm clinical benefits
from introducing molecular studies into pre-operativeSyrenicz et al. Thyroid Research 2014, 7:6 Page 4 of 7
http://www.thyroidresearchjournal.com/content/7/1/6

diagnostic investigations of the thyroid gland as a valu-
able method complementary to ultrasonography and cy-
tology evaluation of thyroid bioptates, particularly whenqualifying patients with follicular adenomas and folli-
cular lesions with undetermined significance for surgical
treatment.
Abbreviations
FNAB: Fine-needle aspiration biopsy; Approx: Approximately; PTC: Papillary
thyroid carcinoma; FTC: Follicular thyroid carcinoma; RNA: Ribonucleic acid;
DNA: Deoxyribonucleic acid; V: Valine; E: Glutamic acid; RET: a proto-
oncogene encoding RTK; TSHR: Thyroid stimulating hormone receptor;CCDC6: Coiled-coil domain-containing gene 6; MAPK: Mitogen-activatedprotein kinase; C: Cytosine; G: Guanine; NCOA4: Nuclear receptor co-activator
4; ELE1: Nuclear receptor co-activator; 4TIMP-3: Tissue inhibitor of
metalloproteinase-3; DAPK: Calcium-dependent protein kinase; SLC5A8:Sodium symporter; NIS: Sodium-iodine symporter; TRbeta: Receptor beta forthyroid hormones; RASSF1A: Signal protein of mitogen RAS pathway;
PPAR γ: Peroxisome proliferator-activated receptor- γ.
Competing interests
The authors have non-financial competing interests (political, personal,
religious, ideological, academic, intellectual, commercial or any other) todeclare in relation to this manuscript.
Authors ’contributions
Prof AS have made substantial contributions to conception and design,acquisition of data and analysis and interpretation of data; have been also
involved in drafting the manuscript, have given final approval of the version
to be published; MK -have made substantial contributions to conception
and design, acquisition of data and analysis and interpretation of data; havebeen also involved in drafting the manuscript, AC – have been also involved
in drafting the manuscript, AS – participated in the sequence alignment and
drafted the manuscript, ABK – carried out the molecular genetic studies,MP – carried out the molecular genetic studies. All authors read and approvedthe final manuscript.
Acknowledgements
This work was supported by a grant from the Polish Ministry of Science and
Higher Education (no. N N402 466739).
Author details
1Department of Endocrinology, Metabolic Diseases and Internal Diseases,
Pomeranian Medical University in Szczecin, Szczecin, Poland.2Department of
Laboratory Diagnostics and Molecular Medicine, Pomeranian MedicalUniversity, Szczecin, Poland.
3Department of Infectious Diseases and
Hepatology, Pomeranian Medical University, Szczecin, Poland.
Received: 11 April 2014 Accepted: 21 May 2014
Published: 17 June 2014
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doi:10.1186/1756-6614-7-6
Cite this article as: Syrenicz et al. :New insights into the diagnosis of
nodular goiter. Thyroid Research 2014 7:6.
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