REVIEW ARTICLE
Thyroid Fine Needle Aspirate: A Post-Bethesda Update
Shikha Bose, MD and Ann E. Walts, MD
Abstract: The Bethesda system for reporting thyroid cytopathology
formulated in 2007 has standardized reporting of thyroid cytology
specimens and streamlined management algorithms. Although 3 of
the categories (benign, malignant, and nondiagnostic) are stan-
dardized and improved, the remaining 3 (follicular lesion of un-
determined significance, follicular neoplasm, and suspicious for
malignancy) remain fraught with interobserver variability and
uncertainty regarding management algorithms. Recent and on-
going morphologic and molecular studies that aim to resolve these
issues are summarized.
Key Words: thyroid, fine needle aspirate, genetic abnormalities,
BRAF, KRAS, Bethesda
(Adv Anat Pathol 2012;19:160–169)
T hyroid carcinoma is the most common endocrine ma-
lignancy although it comprises only 1% of all cancer
diagnoses. It occurs in all age groups, has a female to male
ratio of 3 to 1, and is rapidly gaining importance in public
health and research into novel therapeutics. In 2012, the
National Cancer Institute estimates 56,460 new cases of
thyroid cancer with 1780 disease-related deaths.1 The
worldwide incidence of thyroid cancer has been increasing
and has almost tripled in the last 30 years.2 This has been
attributed to increased detection after the advent of high-
resolution imaging, increased diagnosis consequent to
widespread use of fine needle aspirate (FNA), and de-
creased stringency in histologic criteria for the diagnosis of
papillary thyroid cancer (PTC).1 Given that relative sur-
vival from thyroid cancer substantially declines after age
65, and that the population older than 65 years of age is
expected to double in the next 20 years, an increase in
deaths from thyroid cancer can be expected unless sig-
nificant improvements in the treatment of metastatic dis-
ease can be achieved.3,4 It is estimated that as many as 7%
of adults have at least 1 palpable thyroid nodule and up to
50% of adults have thyroid nodule(s) detectable by ultra-
sonography.5 Currently FNA provides the most cost-
effective method to diagnose thyroid carcinoma, select/
stratify thyroid nodules for surgical management, and/or
confirm the presence of metastatic thyroid cancer. Low
morbidity, high levels of patient acceptance, inexpensive
equipment, and rapid reporting of results have helped make
FNA a key component in the evaluation of virtually all
thyroid nodules.
From the Department of Pathology and Laboratory Medicine, Cedars-
Sinai Medical Center, Los Angeles, CA.
The authors have no funding or conflicts of interest to disclose.
Reprints: Shikha Bose, MD, Department of Pathology and Laboratory
Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd. Suite
8709, Los Angeles, CA 90048 (e-mail: boses@cshs.org).
Figure 2 can be viewed online in color at http://www.anatomic
pathology.com.
Copyright r 2012 by Lippincott Williams & Wilkins
160 | www.anatomicpathology.com
The Bethesda System for Reporting Thyroid Cytopa-
thology (TBST) was developed to provide uniform termi-
nology and diagnostic criteria for reporting thyroid FNAs
and to relate these cytologic diagnoses to clinical manage-
ment. It was formulated by a multidisciplinary group of
thyroid experts who met in Bethesda, Maryland in October
2007.6 TBST describes 6 categories for the diagnosis and
reporting of thyroid FNAs, each with an assigned “risk of
malignancy” and associated recommendations for clinical
management (Table 1).6,7 TBST terminology was sub-
sequently incorporated into the 2009 revised guidelines of
the American Thyroid Association (ATA) for management
of (patients with) thyroid nodules and differentiated thyroid
cancer.8 Several studies have now shown that implementation
of TBST improves the quality of reporting by decreasing the
number of ambiguous reports, increasing the positive pre-
dictive value (PPV) of malignancy in thyroid glands that are
operated, and decreasing the rates of surgery for benign thy-
roid nodules.9–11 TBST does not appear to have affected the
diagnostic accuracy or the false-positive rates of thyroid FNA
or the frequency of intraoperative consultations.10 Three of
the TBST categories (benign, nondiagnostic, and malignant)
have been widely accepted, and their diagnostic criteria are
straightforward and the recommendations for clinical man-
agement are clear. In contrast, issues/controversies regarding
the 3 remaining categories [follicular lesion of undetermined
significance (FLUS), follicular neoplasm (FN), and suspicious
for malignancy] persist, underscoring the need for further
refinement of TBST.
POSITIVE FOR MALIGNANCY
Since adopting TBST most centers have witnessed a
substantial increase in the PPV of thyroid surgery for ma-
lignancy, malignancy rates now routinely exceed 90%.11
These high PPVs are to a large extent attributable to the
fact that about 80% of thyroid malignancies are PTC and
that most aspirates from PTC exhibit features that have
been well described in the literature and provide a high
sensitivity and a high specificity for the diagnosis of con-
ventional PTC (Fig. 1A). Difficulties in recognizing the
follicular variant of papillary carcinoma (FVPTC) in FNAs
are reflected in the lower sensitivity and specificity of cyto-
diagnosis for this variant.12 Although the overall prog-
nosis for PTC is good, approximately 10% to 15% of PTC
exhibit aggressive behavior with frequent local recurrences
and distant metastases. These tumors are often indis-
tinguishable from well-differentiated PTC in aspirate
smears. Few of the aggressive PTC have 1 or more mor-
phologic attributes that while characteristic can never-
theless be difficult to diagnose in an FNA (eg, tall cells in
the tall cell variant of PTC can be confused with Hurthle
cell lesions or with the so-called columnar cell variant of
PTC). The role of molecular markers in identifying the
more aggressive PTC subsets is currently under intense
investigation.
Adv Anat Pathol  Volume 19, Number 3, May 2012
Adv Anat Pathol  Volume 19, Number 3, May 2012 Thyroid Fine Needle Aspirate: A Post-Bethesda Update

invasion for definitive diagnosis. These tumors present

TABLE 1. The Bethesda System for Reporting Thyroid
Cytopathology
Suggested Categories
Benign
Follicular lesion of
undetermined significance
Follicular neoplasm
Suspicious for malignancy
Malignant
Nondiagnostic
challenges for FNA and TBST and are discussed with the
FLUS, FN, and suspicious for malignancy categories
Alternate Risk of below.
Category(s) Terms Malignancy Medullary carcinomas comprise about 4% of thyroid
0%-5% cancers, arise from the parafollicular C-cells, and are often
Atypia of 5%-15% associated with multiple endocrine neoplasia syndromes.
undetermined Although the tumor cells can exhibit a variety of epi-
significance thelioid, plasmacytoid, and/or spindle cell features and
Rule out neoplasm occasional nuclear pseudoinclusions, the presence of highly
Atypical follicular cellular smears exhibiting features that are not typical of
lesion PTC, appropriate clinical history, and confirmatory im-
Cellular follicular
munostain for calcitonin can provide an accurate diagnosis
lesion
in most cases (Fig. 1B). In a recent study, FNA had a
Suspicious for 15%-30% sensitivity of 83% and a PPV of 100% for the diagnosis of
neoplasm medullary carcinoma.13 The sensitivities and PPVs of FNA
— 50%-75% for the diagnosis of other uncommon thyroid malignancies
— 97%-99% including anaplastic carcinoma, lymphoma, and metastatic
Unsatisfactory — carcinoma ranged from 80% to 100% in that study.

Follicular thyroid carcinomas (FTC) comprise 5% to BENIGN

15% of thyroid cancers and, along with the other follicular- TBST has had a major impact on the management of
patterned lesions of the thyroid, require histologic exami- benign lesions of the thyroid as evidenced in the marked
nation to assess capsular integrity and lymph-vascular decrease in the percentage of benign glands excised.9,10

FIGURE 1. Fine needle aspirates showing (A) papillary thyroid carcinoma: monolayerd sheet of atypical follicular cells with nuclear
grooves and intranuclear pseudoinclusions. B, Medullary carcinoma: clusters and single plasmacytoid cells. C and D, Microfollicular
pattern and atypical follicular cells: sheet of atypical follicular cells with crowded and overlapping nuclei, few irregular nuclear mem-
branes, and a rare possible nuclear groove(s).

r 2012 Lippincott Williams & Wilkins www.anatomicpathology.com | 161

Bose and Walts
Accurate cytologic diagnosis of most lesions in the
“benign” category is usually not problematic. However,
the presence of focal Hurthle/Hurthle-like change in the
absence of Hashimoto thyroiditis and the unequal and
heterogeneous distribution of the Hurthle cell and/or lym-
phoid component(s) in Hashimoto thyroiditis can make
it difficult to distinguish chronic thyroiditis and focal
Hurthle-like change in nodular goiter (each categorized as
benign by TBST) from Hurthle cell neoplasm (categorized
as FN by TBST). Although it is understood that the risk of
malignancy for a benign diagnosis by thyroid FNA is im-
pacted by the percentage of benign nodules that are excised,
this level of risk is estimated at <5%.6,7,14
NONDIAGNOSTIC
The inclusion within TBST of precise requirements for
FNA adequacy (at least 6 well-preserved and well-stained
follicular epithelial cell groups, each containing at least 10
cells) and the absence of a “negative for malignancy” cat-
egory (that could encompass aspirates diagnosed as benign
and those that are nondiagnostic) emphasize that “benign”
and “nondiagnostic” are 2 distinct diagnoses in the Be-
thesda system, each with its own recommendations for
follow-up. This clarification has decreased the frustration
previously experienced by clinicians attempting to translate
the variable and often convoluted phrasing that appeared in
earlier reports into patient management. Excluding FNAs
performed by inexperienced aspirators, the majority of
nondiagnostic thyroid aspirates come from nodules that are
either cystic, <5 mm, difficult to palpate, and/or located in
the posterior aspect of the gland. Accordingly, ATA
guidelines8 encourage the use of ultrasound guidance for
aspirations of difficult to palpate nodules and nodules with
>25% cystic component. The guidelines also emphasize
the importance of sampling the solid component of these
mixed (solid and cystic) lesions as a means to reduce the
incidence of nondiagnostic and false-negative FNAs.
After a nondiagnostic FNA, repeat aspiration utilizing
ultrasound guidance has been reported to yield a diagnostic
FNA in as many as 75% of solid nodules and 50% of cystic
nodules.15 In another study, a second benign FNA reduced
the overall false-negative rate of thyroid FNAs from 10.2%
to 4.5%.16 Noting that 90% of their cases with a false-
negative FNA showed increased vascularity or suspicious
features on ultrasound, these authors echo the ATA in
recommending correlation with sonography as a means of
prioritizing nodules that yield nondiagnostic FNAs for re-
aspiration. It is also well known that on-site assessment of
aspirate adequacy substantially decreases the incidence of
nondiagnostic FNAs.16–18 To date, the time-consuming
nature of on-site adequacy assessment has prohibited its
implementation in some cytology laboratories. However, it
is expected that continued advances in telepathology will
help make this service available to many more aspirators in
the near future. About 7% of thyroid nodules (including
some that are malignant on excision) remain nondiagnostic
on repeat aspirations.19,20
FOLLICULAR LESION OF UNDETERMINED
SIGNIFICANCE/FOLLICULAR NEOPLASM/
SUSPICIOUS FOR MALIGNANCY
These 3 TBST categories, collectively referred to as
“indeterminate diagnoses,” comprise from 5% to 42% of
thyroid FNAs, and consist mainly of “follicular-patterned
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Adv Anat Pathol  Volume 19, Number 3, May 2012
lesions.” Diagnosis is on the basis of the relative proportion
of follicular cells with or without atypia, colloid, and a
microfollicular pattern. Using the current Bethesda system,
FNAs from a variety of follicular-patterned lesions (histo-
logically confirmed as hyperplastic nodules, follicular ad-
enomas, FTC, and/or FVPTC) can be diagnosed as FLUS,
FN, or suspicious for malignancy. Inconsistent application
of subjective and overlapping criteria for FLUS, FN, and
suspicious for malignancy is reflected in low levels of in-
traobserver and interobserver diagnostic agreement within
and across these categories and in differences in the risk of
malignancy reported for each category by different labo-
ratories.11,21–26 FLUS, with a TBST assigned 5% to 15%
risk of malignancy, is the most frequent abnormal diagnosis
rendered in thyroid FNAs. Although it is recommended
that FLUS does not exceed 7% of a pathologist’s or a
laboratory’s thyroid FNA diagnoses,6,27 this reported per-
centage varies from 3.0% to 29% across laboratories and
from 2.5% to 28.6% across cytopathologists.11,21,24,28 FN,
which has an assigned 15% to 30% risk of malignancy, is
the second most frequent abnormal diagnosis rendered in
thyroid cytology.
Histologic evaluation is traditionally considered as the
gold standard against which FNA cytology is compared.
The difficult task of refining TBST to reduce the number of
indeterminate thyroid FNAs and to improve the cytohis-
tologic correlation of follicular-patterned lesions is further
complicated by the substantial variability in intraobserver
and interobserver histologic evaluation of follicular-pat-
terned lesions and controversy regarding the criteria that
best define vascular invasion.29 In a study where 6 “experts”
in surgical pathology of the thyroid reviewed sections from
15 follicular-patterned lesions, the intraobserver diagnostic
agreement ranged from 17% to 100% and the unanimous
interobserver agreement on benign and malignant diag-
noses was as low as 27%.30
Review of the recent cytology literature indicates that
several approaches are being explored to increase the clin-
ical utility of TBST with respect to these indeterminate
cytodiagnoses. These approaches involve 1 or more of the
following: (a) reducing the number of diagnostic categories
in TBST by combining or deleting categories in the current
system; (b) determining whether cytologic/nuclear or ar-
chitectural features are more predictive of malignancy and
quantitating the amount or extent of a “diagnostic” feature
that should be required for inclusion in the suspicious for
malignancy category; (c) assessing the value of repeat as-
pirations in indeterminate nodules; (d) requiring consensus
review before diagnosing a thyroid FNA in one of the in-
determinate categories; (e) assessing the potential role of
molecular studies as adjuncts to cytodiagnosis.
(A) Reduce the Number of Diagnostic Categories
in The Bethesda System for Reporting Thyroid
Cytopathology
In a retrospective study using TBST, we identified
considerable overlap in the diagnosis and in the assigned
malignancy risk estimates for the FLUS and FN categories
and for the suspicious for malignancy and malignant cate-
gories and proposed a simplified version of the Bethesda
System for reporting thyroid FNAs that provided 4 non-
overlapping, statistically significant, and more clinically rel-
evant diagnostic categories: unsatisfactory/nondiagnostic,
benign, FLUS/FN, and suspicious for malignancy/malig-
nant.31 Using this 4-category simplified version of TBST
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Adv Anat Pathol  Volume 19, Number 3, May 2012
intraobserver and interobserver diagnostic agreement im-
proved.32 However, in a study of 40 cases originally reported
as FLUS by 2 “experienced” pathologists and reclassified as
benign, FN, suspicious for malignancy, or malignant, Shi
et al,33 found that elimination of the FLUS category de-
creased the sensitivity of thyroid FNA for detecting PTC
from 100% to 27%, increased the false-positive and false-
negative rates for detecting cancer, and decreased intra-
observer and interobserver diagnostic agreement. Tissue
follow-up revealed 37% of the reclassified as benign cases to
be PTC and 38% of the reclassified as FN and suspicious
cases to be benign. The authors concluded that the FLUS
category should not be eliminated although they advocated
minimizing its use. In a multi-institutional study involving the
elimination of the FLUS category and retrospective re-
classification of FNAs originally reported as FLUS, no sig-
nificant differences were observed in the negative predictive
value for the benign category or in the PPV for the FN and
malignant categories when the 5- and 6-category systems
were compared.34 In that study the most significant differ-
ences between the 5-tiered and the 6-tiered systems were the
percentage of cases classified as benign and as FNs. Singh
and Wang35 suggest that the FLUS category could be elim-
inated without decreasing the PPV of the remaining TBST
categories because aspirates with scant cellularity and/or
preparation artifact are often misdiagnosed as FLUS rather
than as nondiagnostic.
(B) Assess the Importance of Cytologic Versus
Architectural Features as Predictors of
Malignancy and Quantitate the Amount or
Extent of a Diagnostic Feature That Should be
Required for Inclusion in the Suspicious for
Malignancy Category
Attempts to determine whether architectural atypia
(microfollicular pattern) (Fig. 1C) or atypical nuclear/
cytologic features (Fig. 1D) are more predictive of malig-
nancy have not been conclusive. In a study that evaluated
the predictive value of 24 cytomorphologic features for
“neoplasia” (defined as adenoma or malignancy in excised
lesions) in FNAs that were originally diagnosed as FLUS,
several nuclear/cytologic features (irregular nuclear mem-
branes, nuclear overlapping, coarse chromatin) and several
architectural features (syncytial tissue fragments, isolated
microfollicles, follicles with scalloped borders) were each
positively correlated with “neoplasia,” whereas honey-
combing, colloid, and histiocytes were inversely correlated
with “neoplasia.”36 In another study of FLUS, cases sub-
classified into 5 groups using combinations of nuclear/
cytologic and architectural atypia, Renshaw37 concluded that
nuclear/cytologic and architectural features are both im-
portant risk factors for malignancy and that microfollicles,
even in the absence of cytologic atypia, should be reported
as FLUS. He suggests that aspirates with cytologic atypia
alone, scant aspirates with so-called “atrophic” follicles,
and cellular aspirates with a mix of macrofollicles and mi-
crofollicles without significant cytologic atypia have similar
risks of malignancy. In contrast, Abele and Levine38 sug-
gested that microfollicles and cytologic atypia (ie, nuclear
enlargement) should both be required for a diagnosis of
FLUS and that in the absence of significant cytologic atypia
(ie, nuclear enlargement), FNAs with microfollicles should
be diagnosed as benign. VanderLaan et al39 found that
FNAs with cytologic atypia, both cytologic and architec-
r 2012 Lippincott Williams & Wilkins
Thyroid Fine Needle Aspirate: A Post-Bethesda Update
tural atypia (microfollicles), or “unspecified” atypia carried
twice the risk of malignancy as compared with FNAs ex-
hibiting architectural atypia alone. They noted that 90% of
the cancers that followed a diagnosis of FLUS were PTC of
which 85% were FVPTC. Architectural atypia alone was
more likely to predict follicular adenoma than PTC. In a
retrospective review of FNAs diagnosed as “suspicious for
follicular neoplasm (FN),” Lubitz et al40 calculated the
predicted probability of malignancy as 88.4% when the
nodule was Z4 cm and nuclear grooves and a transgressing
vessel were both identified in the aspirate. If the authors
excluded cases diagnosed as PTC on histology from the
study, anisokaryosis and presence of nucleolus replaced the
presence of nuclear grooves as significant predictors of
malignancy, and then nodule size (Z4 cm), a transgressing
vessel and anisokaryosis lacking a nucleolus had a predicted
probability of malignancy of 96.5%.
Attempts to quantitate cytologic and architectural
features that are qualitative and subjective are fraught with
potential errors and have poor reproducibility. In a recent
study designed to determine whether cytologic or archi-
tectural atypia could further stratify FLUS to predict car-
cinoma, we eliminated the confounding effects of subjective
and semiquantitative terms such as “rare,” “few,” “some,”
“focal,” “probable,” “possible” which were used differently
across cytopathologists and scored each feature on a binary
scale (present or absent) with possible and probable con-
sidered as present. In our review of 83 consecutive FNAs
diagnosed as FLUS and subsequently excised, comparison
of the FNAs from the benign and malignant cases showed
significant differences (P < 0.05) in the frequencies of nu-
clear grooves/irregular nuclear membranes, nuclear over-
lap/crowding, nuclear pseudoinclusions, and Hurthle
change. The presence of nuclear overlap/crowding was
strongly correlated with the presence of nuclear grooves/
irregular membranes, and nuclear pseudoinclusions and
malignancy was present in 61% of the cases whose FNAs
exhibited one or more of these features indicating that these
FNAs are more appropriately diagnosed as suspicious for
malignancy (the category to which TBST assigns a risk of
malignancy of 50% to 75%). No significant differences
were observed in the frequencies of cell sheets, micro-
follicles, nuclear enlargement, colloid, lymphocytes, bland/
compact chromatin, or cell aggregates/clusters reported in
aspirates from the benign and malignant cases.41
(C) Assess the Role of Repeat Fine Needle
Aspirate After an Indeterminate Diagnosis on
Fine Needle Aspirate
Although there is general agreement that repeat FNA
is helpful in the clinical management of FLUS cases, repeat
FNA is generally not recommended for nodules diagnosed
as FN or suspicious for malignancy as these nodules are
best excised.22,37
(D) Consensus Review as a Prerequisite for a
Diagnosis of Follicular Lesion of Undetermined
Significance
By conducting a group consensus review of 50 thyroid
aspirates originally reported as FLUS, Jing et al42 achieved
a 78% reduction in the number of FNAs reported as
FLUS, provided optimal interobserver diagnostic agree-
ment, and substantially improved cytohistologic con-
cordance. In the study, approximately 50% of the cases
originally reported as FLUS were reclassified as benign.
www.anatomicpathology.com | 163
Bose and Walts Adv Anat Pathol  Volume 19, Number 3, May 2012

Although the ability of group consensus review to improve

diagnostic accuracy is recognized, this activity is time
consuming and not feasible for a large number of multislide RAS
cases on a routine basis. PAX8/PPAR BRAF
Follicular cells
(E) Molecular Studies as Adjuncts
Because differences in case selection, study design, and Well differentiated Well differentiated
other confounding factors make it difficult to compare/ Follicular Ca 15% Papillary Ca 80%
reconcile results from morphology-based studies and/or to
reach definitive conclusions on how best to improve the
diagnosis and management of cases that yield indeterminate
cytologic diagnoses, molecular technologies (genomics,
proteomics, and metabolomics) are actively being explored Poorly differentiated
as potential diagnostic, therapeutic, and prognostic ad- Ca
juncts to thyroid FNA.

+ +
GENETIC ALTERATIONS IN NONMEDULLARY TP53
THYROID CANCER TP53
CTNNB1 CTNNB1
The initiation and progression of nonmedullary thyroid
PIK3CA PIK3CA
cancer involves the accumulation of various genetic and ep-
AKT1
igenetic alterations. Although hereditary forms of PTC are AKT1
known, definite susceptibility genes have not been defined.43 PTEN Anaplastic Ca PTEN
In contrast, FTC is known to be associated with some fam-
ilial syndromes (Table 2).44–48 Familial nonmedullary thyroid
carcinomas are heterogeneous diseases, with early age onset,
are usually bilateral and multicentric, and comprise about
10% to 15% of thyroid cancers.43 The sporadic forms of
FIGURE 2. Nonmedullary thyroid carcinogenesis
thyroid cancer are commonly initiated by mutations in ef-
fector genes (RET, BRAF, RAS) of the mitogen-activated
protein kinase (MAPK) pathway49 while progression occurs
substitution occurs at residue 1799 in >95% of mutations
by alterations in the phosphatidylinositol 3 (PI3)-kinase-
resulting in a valine to glutamate replacement at position
AKT pathway (Fig. 2). Point mutations and chromosomal
600.49,51 BRAF mutations are detected in 40% to 45% of
rearrangements are the most common mechanisms of genetic
PTCs with the highest incidence reported in the tall cell
alteration. Point mutations, commonly seen in BRAF and
(80%) and classic (60%) variants. BRAF mutations are
RAS genes, involve the substitution of a single nucleotide
infrequent in FVPTC (10%) and in PTC that occur in
within the DNA chain and result in an altered protein that is
young individuals and/or are associated with radiation (0%
constitutively activated. These mutations have been found to
to 12%).52–54 Although BRAF mutation is reported to be
be mutually exclusive.50
specific for PTC, a low rate of false positivity has been
Chromosomal rearrangements, as observed with RET/
reported in studies from Korea.55,56 BRAF mutations are
PTC and PAX8/PPARg, occur as a result of breakage and
also identified in 20% to 40% anaplastic carcinoma.57,58
transference of a segment of 1 chromosome to a new site on
Several studies have confirmed the association of
the same chromosome or to a nonhomologous chromo-
BRAF mutation with aggressive tumor behavior.59–61 In a
some. This transfer either approximates the promoter of 1
recent meta-analysis of 27 studies (total, 5655 patients), the
gene to the coding sequence of the second gene (RET/PTC)
incidence of BRAF mutation was 49% and BRAF mutation
leading to excessive production of the protein or approx-
was associated with an increased likelihood of extra-
imates 2 coding sequences resulting in the formation of a
thyroidal extension [odds ratio (OR), 2.14; 95% confidence
fusion protein (PAX8/PPARg). These proteins then func-
interval (CI), 1.68-2.73], lymph node metastasis (OR, 1.54;
tion as oncogenes that drive the MAPK pathway.
95% CI, 1.21-1.97), and advanced tumor stage (OR, 2.00;
95% CI, 1.61-2.49). Eight of the studies showed 2-fold
BRAF MUTATIONS increases in the risk of recurrent/persistent disease (95%
BRAF is an intracellular effector of the MAPK path- CI, 1.67-2.74).60 BRAF mutation has also been associated
way. In BRAF V600E, a thymine to adenine nucleotide with an increased incidence of tumor-related mortality.62

TABLE 2. Familial Syndromes Associated With Thyroid Carcinomas of Follicular Origin

Familial Syndrome Characteristics Gene Mutated (Location) Incidence of Carcinoma
Cowden syndrome Mucocutaneous hamartomas benign and malignant tumors PTEN (10q23) 5%-10%
of the breast, endometrium, and thyroid
Werner syndrome Premature aging (bilateral cataracts, gray hair, skin atrophy) WRN (8p11-p12) 15%-20%
benign and malignant thyroid lesions
Carney complex type 1 Pigmented skin and mucosal lesions variety of endocrine PRKARIA 15%
neoplasias

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Adv Anat Pathol  Volume 19, Number 3, May 2012
Studies are in progress to determine whether optimization
of surgical treatment of BRAF-associated PTC will be
beneficial.63
RET/PTC TRANSLOCATION
The RET/PTC translocation involves the trans-
location of the kinase domain of the RET gene located on
chromosome 10q11.2 to the promoter of at least 15 differ-
ent genes located on chromosome 10 or on various other
chromosomes, resulting in aberrant ligand-independent
activation of the MAPK pathway.50 The most common
rearrangements are the RET/PTC1 (inv 10)(q11.2;q21.2)
and RET/PTC3 (inv 10)(q11.2;q11), both of which are
paracentric intrachromosomal inversions.64,65 The dis-
tribution of the RET/PTC rearrangement is heterogeneous.
Clonal rearrangements occurring in >1% of tumor cells
are exclusively identified in 10% to 20% of PTC.66 These
mutations are commonly found in PTC of younger in-
dividuals or children and are associated with ionizing ra-
diation.67 RET/PTC1 is seen in PTC exhibiting classic
morphology whereas RET/PTC3 is commonly observed in
the solid variant. Sensitive methods have also detected
nonclonal rearrangements in <1% of neoplastic cells in
10% to 45% of benign lesions and adenomas.68–70
RAS MUTATIONS
Activating point mutations are located in NRAS,
HRAS, and KRAS at codons 12, 13, and 61, with NRAS
and HRAS codon 61 mutations being the most common.71,72
RAS mutations are found in follicular-patterned thyroid le-
sions including 40% to 50% of FTC, 20% to 40% of ana-
plastic carcinomas, and 10% to 20% of FVPTC.73,74 RAS
mutations are also found in 20% to 40% of follicular ad-
enomas and in up to 20% of adenomatous goiters raising
the possibility that these lesions might represent precursor
lesions.74,75
PAX8/PPARg
PAX8/PPARg translocation is identified in follicular-
based lesions and involves an in-frame fusion of PAX8
(thyroid transcription factor) with PPARG1 (peroxisome
proliferator-activated receptor-g 1) gene, t(2;3)(q13;p25)
resulting in overexpression of a fusion protein.76 The
mechanism of oncogenesis is not yet clear. This re-
arrangement is identified in 20% to 70% of FTC, 0% to
20% of follicular adenomas, and 0% to 5% of PTC.77–81
Tumors with this rearrangement are associated with a mi-
crofollicular, solid, or trabecular growth pattern, a thick
fibrous capsule and overexpression of galectin-3 and/or
HBME-1, markers commonly used in distinguishing benign
from malignant thyroid tumors. This fusion mutation has
not been reported in goiters or poorly differentiated/ana-
plastic carcinomas.
ADDITIONAL ALTERATIONS
Mutations involving TP53, CTNNB1 (b-catenin), and
less frequently various genes in the PI3 kinase signaling
pathway (PI3CA, PTEN, and AKT1) accumulate in poorly
differentiated and anaplastic carcinomas as late events and
are thought to be indicative of dedifferentiation and pro-
gression.82–86
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Thyroid Fine Needle Aspirate: A Post-Bethesda Update
MicroRNA SIGNATURES
MicroRNAs are small noncoding RNAs that function
as negative regulators of protein expression. Many micro-
RNAs have been found to be deregulated in thyroid cancer
and several such as miR-146b, miR-221, and miR-222 are
highly upregulated in PTC, whereas others are abnormally
expressed in FTC (miR-197, miR-346, miR-155, and miR-
224) and anaplastic carcinomas (miR-30d, miR-125b, miR-
26a, and miR-30a-5p). Although the role of these micro-
RNAs in tumorigenesis is currently unclear, studies are in
progress to determine their utility in the diagnosis and
prognosis of thyroid cancer.87–92
CLINICAL UTILITY OF MOLECULAR MARKERS
Thyroid FNA is an accurate test with good positive
and negative predictive values for malignant and benign
diagnoses. However, in cases where FNA results are in-
determinate, the revised ATA management guidelines rec-
ommend that clinicians consider molecular testing to help
guide clinical management.8
Because of its relative frequency and specificity for
PTC, BRAF has been the most common gene tested in
FNA samples. A meta-analysis of 22 such studies50 revealed
that FNA material is reliable for molecular analysis and
that the V600E mutation is highly specific for PTC. A total
of 99.3% (1109/1117) of the BRAF-positive nodules were
PTC on final histopathology. Five of the 8 false-positive
cases (no cancer identified by histology) were reported in 1
study that used a more sensitive detection method. Thus,
BRAF mutation positivity confers >99% risk of malig-
nancy. In this study, 15% to 40% of the BRAF-mutated
samples had an indeterminate FNA cytology result, in-
dicating that the presence of BRAF mutation might be of
diagnostic value in these cases.
As BRAF mutations occur in only 40% to 45% of
PTC, testing for this mutation alone would miss a sub-
stantial number of PTC and other BRAF-mutation–neg-
ative thyroid cancers. Several studies have investigated the
usefulness of a panel of mutations including BRAF and
RAS mutations and RET/PTC and PAX8/PPARg re-
arrangements for analysis of thyroid FNA samples.93–97
These studies demonstrate that in an appropriate clinical
setting the presence of any mutation is a strong predictor of
malignancy in thyroid nodules irrespective of the cytologic
diagnosis.93–95 The presence of BRAF, RET/PTC, or
PAX8/PPARg correlates with the presence of malignancy
in 100% of cases, whereas RAS mutations have a 74% to
87% PPV for cancer, consistent with the known presence of
RAS mutations in some benign follicular adenomas and
goiters. Despite the lower specificity for malignancy, RAS
mutations have the attribute of being positive in more
challenging cytologic diagnoses such as FVPTC and FTC.
A recent prospective analysis98 of residual material
from 1056 consecutive thyroid FNA samples with in-
determinate cytology (ie, Bethesda categories of FLUS,
FN, and suspicious for malignancy) showed that the re-
sidual material was adequate for molecular analysis in 92%
of cases. A total of 479 of the patients underwent surgery.
The mutation analysis was performed using a newly de-
veloped polymerase chain reaction assay for a panel of
mutations: BRAF V600E, NRAS codon 61, HRAS codon
61, and KRAS codons 12/13 point mutations and RET/
PTC1, RET/PTC3, and PAX8/PPARg rearrangements and
included stringent assessment for the presence of epithelial
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Bose and Walts
cells. Detection of any mutation conferred 88%, 87%, and
95% risk of malignancy in the FLUS, FN, and suspicious
for malignancy Bethesda categories, respectively. The risk
of cancer in mutation-negative nodules was 6%, 14%, and
28% in the FLUS, FN, and suspicious for malignancy
categories, respectively. There were 13 mutation-negative
cancers in the FLUS FNAs. Of these, only 1 lesion showed
extrathyroidal extension. It is noteworthy that even with
the use of this panel of mutations, substantial numbers of
thyroid cancers in the FN and suspicious for malignancy
groups remained mutation negative. Although the clinical
management of these patients was not significantly affected,
Nikiforov et al98 proposed that these patients can be offered
lobectomy instead of the 1-step total thyroidectomy, which
is appropriate in mutation-positive cases.
Although molecular testing is reported to decrease the
false-negative rate of benign cytologic diagnoses, there is
currently little impetus to perform molecular analysis on
these samples as the risk of malignancy is <5%. Various
studies have reported reductions in false-negative rates in
FNAs diagnosed as benign from 2.1% to 0.9%95 and 10%
to 6%.94 The cost-effectiveness of testing all negative cy-
tology samples and the potential benefit from the detection
of only a few cancers requires further investigation.
Similarly, it is not established whether molecular testing
of FNA specimens diagnosed as malignant should be under-
taken. BRAF-mutated tumors may benefit from more ex-
tensive initial surgery as it has been shown that, because of the
increased incidence of cervical recurrence, these tumors un-
dergo more frequent reoperation than tumors without the
mutation.63,99 Thus, Yip et al63 suggest that knowledge of
BRAF mutational status might optimize the extent of surgery
and lymph node dissection. In addition, on the basis of data
that suggest BRAF-mutated PTC are more resistant to radio-
iodine treatment because of a reduced ability to trap radio-
iodine,100,101 an increased dose of radioiodine, less suppression
of the thyroid stimulating hormone, and closer follow-up has
been suggested as initial postoperative treatment of BRAF-
mutation–positive cancers.102 Knowledge of the mutation sta-
tus may also provide options for targeted therapy.103
The association between BRAF V600E and aggressive
disease characteristics has also been reported in papillary
microcarcinomas (tumorsr1 cm).104–107 Although BRAF
mutation is identified in 24% to 63% of papillary micro-
carcinomas, <10% to 15% of these tumors behave ag-
gressively. Howell et al108 reported that tumor recurrence is
limited to older (65 years and above) patients. Using BRAF
mutation status, a set of 3 pathologic features (superficial
tumor location, intraglandular tumor spread/multifocality,
and tumor fibrosis), and stepwise regression analysis, Nie-
meier et al109 developed a combined molecular pathologic
score for predicting aggressive tumor behavior. Using this
score, their sensitivity and specificity for detection of ex-
trathyroidal spread or recurrence increased from 77% to
96% and from 68% to 80%, respectively; thus indicating
that BRAF mutational status could stratify papillary mi-
crocarcinomas for aggressive clinical management.
CONCLUSIONS
The Bethesda classification has standardized reporting
of thyroid cytology specimens and streamlined manage-
ment algorithms. However, it has generated considerable
controversy regarding the use and management of the in-
determinate category. Many studies (both morphologic and
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Adv Anat Pathol  Volume 19, Number 3, May 2012
molecular) are ongoing in an effort to improve the diag-
nosis and management of this group of cases. Whether
molecular analysis alone or in concert with morphologic
evaluation will provide reliable stratification of in-
determinate thyroid FNAs is not yet clear. Cost-effective-
ness, management algorithms, and specific indications will
also need to be determined.
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