Florida International University
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HWCOM Faculty Publications Herbert Wertheim College of Medicine
2017
Genomic Pro&ling of %yroid Nodules: Current
Role for %yroSeq Next-Generation Sequencing
on Clinical Decision-Making
Atil Y. Kargi
University of Miami Hospital
Marcela Perez Bustamante
University of Miami Hospital
Seza Gulec
Herbert Wertheim College of Medicine, Florida International University, sgulec@=u.edu
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Recommended Citation
Kargi, Atil Y.; Perez Bustamante, Marcela; and Gulec, Seza, "Genomic Pro=ling of <yroid Nodules: Current Role for <yroSeq Next-
Generation Sequencing on Clinical Decision-Making" (2017). HWCOM Faculty Publications. 114.
h>ps://digitalcommons.=u.edu/com_facpub/114
Review
Mol Imaging Radionucl Ther 2017;26(Suppl 1):24-35 DOI:10.4274/2017.26.suppl.04
24
Ad dress for Cor res pon den ce: Atil Y. Kargi MD, University of Miami Hospital, Clinic of Diabetes and Metabolism, Division of Endocrinology, Florida, USA
Phone: 305-243-3636 E-mail: akargi@med.miami.edu
Atil Y. Kargi
1
, Marcela Perez Bustamante
1
, Seza Gulec, MD, FACS
2
1
University of Miami Hospital, Clinic of Diabetes and Metabolism, Division of Endocrinology, Florida, USA
2
Florida International University Herbert Wertheim College of Medicine, Departments of Surgery and Nuclear Medicine, Miami, USA
Genomic Profiling of Thyroid Nodules: Current Role for ThyroSeq
Next-Generation Sequencing on Clinical Decision-Making
Tiroid Nodülü Genomik Profillemesi: ThyroSeq Yeni-Nesil Dizinlemenin Klinik Karar Verme
Üzerinde Güncel Rolü
Abstract
In recent years there has been an increased awareness of the genetic alterations underlying both benign and malignant
neoplasms of the thyroid. Next-generation sequencing (NGS) is an emerging technology that allows for rapid detection of a
large number of genetic mutations in thyroid fine-needle aspiration (FNA) specimens. NGS for targeted mutational analysis
in thyroid tumors has been proposed as a tool to assist in the diagnosis of thyroid nodules with indeterminate FNA cytology.
Results of genomic testing of thyroid nodules and thyroid cancers could also have prognostic implications and play a role
in determining optimal treatment strategies including targeted therapies. We provide a critical review of existing studies
assessing the performance of the ThyroSeq NGS test for the diagnosis and management of patients with thyroid nodules
with indeterminate cytopathology and discuss the applicability of findings from these studies to clinical practice. While there
are early indications to suggest a possible utility of data obtained from NGS to aid in prognostication and therapeutic decision-
making in thyroid cancer, we recommend judicious use and cautious interpretation of such molecular testing until results
of ongoing clinical trials become available. Lastly, we discuss recommendations provided from clinical practice guidelines
regarding the use of mutation detection via NGS in the diagnostic evaluation of thyroid nodules.
Keywords: Thyroid cancer, thyroid nodule, genomic profiling, next-generation sequencing, ThyroSeq, molecular testing
Öz
Son yıllarda benign ve malign tiroid neoplazmlarının altında yatan genetik değişiklerle ilgili artan bir farkındalık oluşmuştur.
Yeni nesil dizinleme [Next-generation sequencing (NGS)] tiroid ince iğne aspirasyon (İİA) örneklerinde çok sayıda genetik
mutasyonun hızlı tespitine izin veren ve gelişmekte olan bir teknolojidir. Tiroid tümörlerinin hedeflenen mutasyon analizi
için NGS, İİA ile belirsiz sitoloji saptanan tiroid nodüllerinin tanısında yardımcı olmak için bir araç olarak öne sürülmüştür.
Ek olarak, tiroid nodülü ve tiroid kanserlerinin genomik test sonuçlarının prognostik etkileri olabilir ve hedefli tedaviler dahil
olmak üzere uygun tedavi stratejilerinin belirlenmesinde önemli bir rol oynayabilir. Bu yazıda İİA ile belirsiz sitolojisi olan tiroid
nodüllü hastaların tanı ve tedavisinde ThyroSeq NGS testinin performansını değerlendiren mevcut çalışmaların bir derlemesini
sunmayı ve bu çalışmalardan elde edilen bulguların klinik pratikte uygulanabilirliğini tartışmayı amaçladık. Her ne kadar erken
veriler NGS’den elde edilen verilerin tiroid kanserinde prognoz ve tedavi kararı sürecine olası yardımlarını öne sürse de, bu tür
moleküler testlerin devam eden klinik çalışmaların sonuçları belirlenene kadar, akıllıca kullanılmasını ve dikkatli yorumlanmasını
öneriyoruz. Son olarak, tiroid nodüllerinin tanısal değerlendirilmesinde NGS ile mutasyon tespiti ile ilgili klinik uygulama
kılavuzlarından edinilmiş öneriler tartışılmıştır.
Anahtar kelimeler: Tiroid kanseri, tiroid nodülü, genomik profilleme, yeni nesil dizinleme, ThyroSeq, moleküler test
©
Copyright 2017 by Turkish Society of Nuclear Medicine
Molecular Imaging and Radionuclide Therapy published by Galenos Yayınevi.
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Kargi et al. ThyroSeq Genomic Profiling of Thyroid Nodules Role in Clinical Decision-MakingMol Imaging Radionucl Ther 2017;26(Suppl 1):24-35
Introduction
Thyroid nodules are common in the general population
with higher prevalence in women and in older persons.
When ultrasound is performed at random in the general
population 19-68% of individuals are found to harbor one
or more thyroid nodules (1). While the majority of these
nodules are not clinically significant 7-15% are malignant
(2). Paralleling the increased use of imaging techniques
and of thyroid fine-needle aspiration (FNA) there has been
dramatic increase worldwide in both the incidence of
thyroid nodule diagnosis and that of thyroid cancer over
the past 20-30 years (3,4).
Several clinical practice guidelines have set forth strategies
to manage patients who are discovered to have thyroid
nodules, yet a great deal of controversy still exists as to
the optimal approach to diagnosis and treatment (5,6).
The widespread use of high-resolution ultrasonography of
the neck as well as thyroid FNA has significantly enhanced
our ability to diagnose malignancy among thyroid nodules,
however 20-30% of cytology results from thyroid FNA
fall into one of three indeterminate diagnostic categories
according to the Bethesda System for Reporting Thyroid
Cytopathology: Atypia of undetermined significance/
Follicular lesion of undetermined significance (AUS/FLUS)
(Bethesda category III), follicular neoplasm/suspicious for
follicular neoplasm (FN/SFN) (Bethesda category IV), and
Suspicious for malignancy (SM) (Bethesda category V) (7).
The reported frequency and risk of malignancy with each of
the Bethesda reporting categories is summarized in Table 1.
Patients and physicians faced with an indeterminate
cytopathology report will have to make the sometimes
difficult decision of deciding on the next step in
management of the thyroid nodule, which until recent
years has meant choosing from one of three options: repeat
FNA, observation with continued ultrasound surveillance or
surgical management. Each of these strategies brings with
it specific considerations and complexities; for instance
in patients referred for surgery the need to decide upon
the extent of thyroidectomy and the potential need for
a two-step procedure of thyroid lobectomy followed by
a completion thyroidectomy in the circumstance that the
lobectomy results in a diagnosis of thyroid cancer.
Taking into consideration that many thyroid cancers
are indolent tumors and that many patients may have
an excellent prognosis even when the diagnosis and
treatment has been delayed and the fact that most patients
who undergo thyroidectomy for AUS/FLUS and FN/SFN
cytopathology will be diagnosed with benign nodules on
final surgical histopathology, clinicians and patients have
been left with weighing the risks of a more conservative
strategy of surveillance with that of the more aggressive
approach of proceeding to thyroidectomy.
A variety of factors can predict the risk of cancer and aid in
the decision on optimal management for patients presenting
with nodules having indeterminate cytopathology; including
patient risk factors (age, gender, family history, past exposure
to ionizing radiation), serum TSH level and presence or
absence of sonographic features suspicious for papillary
thyroid cancer (PTC) (6,8,9). In their 2015 management
guidelines pertaining to adults with thyroid nodules, the
American Thyroid Association (ATA) has provided clear
guidance on the criteria that should be used to determine
the initial indication for FNA based on traditional risk factors
and in particular a risk stratification model heavily reliant
upon sonographic appearance of the nodule (6).
However, in the circumstance that FNA is performed,
once patients and health care providers are faced with
indeterminate cytology, it becomes much less clear from the
guidelines precisely how the same criteria should be used
to inform management decisions. This situation has created
a need to improve on the cytological inaccuracy inherent
to the diagnosis of indeterminate thyroid FNA, resulting in
the development of a number of new diagnostic modalities
intended for application as a “rule-in” or “rule-out” test for
thyroid cancer. When discussing the performance of any
of these tests it must be taken into consideration that the
ideal ‘rule-in’ test should have a positive predictive value
(PPV) similar to that of a malignant cytological diagnosis
Table 1. Bethesda system for the classication of thyroid cytopathology
Bethesda
category
Cytopathologic
category
Approximate
expected frequency
Malignancy rate Suggested treatment (Prior to
availability of molecular testing)
I Non-diagnostic/Inadequate 5-11% 1-4% Repeat FNA
II Benign 55-74% 0-3% US follow-up
III Atypia/follicular lesion of
undetermined significance
5-15% 5-15% Repeat FNA or US follow-up or
Lobectomy
IV Follicular neoplasm/
suspicious for FN
2-25% 15-30% Lobectomy
V Suspicious for malignancy 1-6% 60-75% Lobectomy or Thyroidectomy
VI Malignant 2-5% 97-99% Near-total thyroidectomy
FNA: Fine-needle aspiration, FN: Follicular neoplasm, US: Ultrasonographic
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Kargi et al. ThyroSeq Genomic Profiling of Thyroid Nodules Role in Clinical Decision-Making Mol Imaging Radionucl Ther 2017;26(Suppl 1):24-35
(Bethesda category VI) (98.6%), while an ideal ‘rule-
out’ test should have a negative predictive value (NPV)
comparable to that of benign cytology (Bethesda category
II) (96.3%) (7).
In recent years a number of diagnostic tests have been
evaluated to aid in the diagnosis of indeterminate thyroid
nodules, including FDG-PET and several assessments of
molecular markers in FNA specimens (10,11). Molecular
tests include immunohistochemistry for Galectin -3, HBME-
1 and CK19; gene expression and microarray analysis;
microRNA expression; and testing for mutations and gene
rearrangements (6,12). Currently in the U.S.A. commercially
available molecular tests include those for single or multiple
mutation analysis, combination panels for mutation analysis
and chromosomal rearrangements (miRInform®-Asuragen,
ThyroSeq-CBLPath and University of Pittsburgh Medical
Center) and a proprietary gene expression classifier
(Afirma GEC®-Veracyte) (13,14,15). While initially the
gene expression classifier (GEC) was proposed as the best
among these tests to rule-out malignancy and mutation
analysis was preferred as a “rule-in”, the next-generation
sequencer (NGS) ThyroSeq has recently been shown to
have both a high PPV and NPV for thyroid cancer diagnosis
when applied to thyroid FNA with indeterminate cytology
(16,17,18). A further strength of the ThyroSeq, when
compared to GEC, is that it provides detailed and specific
information regarding the exact genetic alteration driving
the disease, which could potentially provide prognostic and
therapeutic implications including impacting upon extent of
surgery, use of RAI and possible future targeted therapies.
Thyroid cancer, like all cancers, is a disease of the genome.
The initiation and progression of cancer is due to the
accumulation of genetic and epigenetic changes such as
somatic mutations, chromosomal rearrangements, micro
RNA dysregulation and alterations in gene expression
(19). In differentiated thyroid cancer (DTC), the observed
genetic changes frequently lead to activation of the
MAPK or PI3K-AKT pathways. Approximately 70% of DTC
demonstrate one of four genetic abnormalities: point
mutations in the BRAF or RAS gene or either one of two
chromosomal rearrangements: RET/PTC or PAX8/PPARG
(19). Our knowledge of the genomic alterations explaining
the remaining approximately 30% of all thyroid cancers
not harboring one of the aforementioned four genetic
aberrations has been greatly expanded by a number of
recent discoveries, including those reported in 2014 by
the National Cancer Genome Atlas Research Network, in
which the genetic driver was identified in 96.5% of 496
PTC cases (20,21). The findings of this last report have led
some experts to propose a reclassification of PTCs based on
molecular characteristics to better reflect their underlying
differentiation and signaling properties (21). While a
detailed discussion of all current molecular tests in thyroid
FNA is beyond the scope of this article, our review will focus
on the role of NGS, a methodology which we believe may
hold particular promise in diagnosis of thyroid FNA as well
as future potential for use in prognostication and informing
management of patients with thyroid cancer.
Next-generation Sequencing in Thyroid Fine-
Needle Aspiration
NGS is a method of simultaneous sequencing of a very
large number of short nucleic acid sequences that can be
used to detect multiple genetic alterations in large regions
of the genome (22). Compared to standard methods of
sequencing, such as Sanger sequencing, NGS has the
advantage of rapid simultaneous sequencing of large
sections of the genome and quantitative assessment
of mutated alleles. NGS can be used for whole-genome
sequencing as well as in a more targeted manner directed
at specific mutations in specific areas of the genome.
The ThyroSeq NGS panel provides simultaneous sequencing
for detection in over a thousand hotspots of 14 thyroid
cancer-related genes and for 42 types of gene fusions
occurring in thyroid cancer (14,17). The genes analyzed for
mutation are AKT1, BRAF, CTTNB1, GNAS, HRAS, KRAS,
NRAS, PIK3CA, PTEN, RET, TP53, TSHR, TERT and EIF1AX.
The gene list for gene fusions and expression consists of
RET, PPARG, NTRK1, NTRK3, ALK, IGF2BP3, BRAF, MET,
CALCA, PTH, SLC5A5, TG, TTF1, KRT7 and KRT20.
The proposed uses for NGS for thyroid FNA samples include
diagnosis of cytologically indeterminate thyroid nodules,
prognostication in thyroid cancer and to inform selection
of targeted therapies (14). The possible applications and
indications of ThyroSeq include:
1. Thyroid FNA with indeterminate cytology (Bethesda
categories III, IV and V),
2. Malignant thyroid cytology (Bethesda category VI),
when results of the NGS are expected to affect the decision
for extent of oncological surgery,
3. Benign thyroid cytology (Bethesda category II), when
strong SM exists on clinical grounds such as presence of a
highly suspicious sonographic pattern,
4. When the diagnosis of thyroid cancer is established
cytologically or histologically and molecular profiling
will effect decision regarding radioactive iodine therapy,
intensity of follow up, or for selection of targeted therapies
in patients with advanced cancer.
We will discuss the potential roles of NGS in thyroid FNA
specimens below, with an emphasis on its role in clinical
decision-making.
Potential Role of Next-Generation Sequencer in
AUS/FLUS (Bethesda Category III) Cytology
The diagnosis of AUS/FLUS should be made in FNA
specimens containing cells with architectural and/or
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Kargi et al. ThyroSeq Genomic Profiling of Thyroid Nodules Role in Clinical Decision-MakingMol Imaging Radionucl Ther 2017;26(Suppl 1):24-35
nuclear atypia more pronounced than expected for benign
changes, yet not sufficient to be classified in one of the
higher risk Bethesda categories (7). Although this diagnosis
has an expected and recommended frequency of 7%,
recent analyses have found this cytological category to be
diagnosed in 1-27% of all thyroid FNA specimens (23). In
studies assessing the risk of cancer in patients with Bethesda
category III nodules, the rate of malignancy diagnosed in
patients who went to surgery was 6-48%, with a mean risk
of 16% (24).
To date only one study has assessed the performance of
ThyroSeq in AUS/FLUS (17). In this study 465 FNA samples
from 441 patients at a single institution diagnosed as AUS/
FLUS on cytology were submitted prospectively to ThyroSeq
molecular testing. In addition to the 42 gene fusions and
14 genes analyzed for point mutation, expression of eight
genes were analyzed to evaluate the cell composition of the
needle aspirates. Ninenty-eight of the cases (21%) had a
definitive diagnosis by either surgical (n=96) or non-surgical
(n=2) methods. Of all FNA samples 462 were determined
to be composed of follicular cells while three samples were
diagnosed as parathyroid in origin. Among the samples
consisting of follicular cells 31 were positive on mutational
analysis (6.7%) (Figure 1).
Of the entire group of 441 patients, 96 nodules occurring
in 90 patients were surgically removed due to the finding
of an additional nodule in the same gland with either
Bethesda V or Bethesda VI cytology in five patients.
Twenty-seven patients underwent thyroidectomy because
of positive ThyroSeq results and the remaining cases were
Figure 1. Schematic representation of study flow and overall performance of ThyroSeq in thyroid nodules with atypia of undetermined significance/
follicular lesion of undetermined significance. Results showed sensitivity 90.0% [confidence interval (CI) 78.8-100], specificity 92.1% (CI 86.0-98.2), positive
predictive value 76.9% (CI 60.7-93.1) and negative predictive value 97.2% (CI 78.8-100) with accuracy of 91.8% (CI 86.4-97.3). The overall prevalence
of a thyroid cancer diagnosis in the study of all samples of follicular cells (n=462) that underwent molecular testing was 4.8%. (Adapted from Nikiforov
YE, Carty SE, Chiosea SI, Coyne C, Duvvuri U, Ferris RL, Gooding WE, LeBeau SO, Ohori NP, Seethala RR, Tublin ME, Yip L, Nikiforova MN. Impact of the
multi-gene ThyroSeq next-generation sequencing assay on cancer diagnosis in thyroid nodules with atypia of undetermined significance/follicular lesion of
undetermined significance cytology. Thyroid 2015;25:1217-1223).