Molecular markers in thyroidcytology: diagnostic andprognostic implicationsExpert Rev. Endocrinol. Metab. 8(5), 439–448 (2013)

Maryam I Khan* andMarc J LaufgrabenDivision of Endocrinology, Diabetes and

Metabolism, Cooper Medical School of

Rowan University, Cooper University

Health Care, Camden, NJ 08103, USA

*Author for correspondence

Tel.: +1 8563422312

Fax: +1 8569660735

khan-maryam@cooperhealth.edu

The discovery of thyroid nodules in the general population has risen markedly with thegreater use of ultrasound resulting in increasing use of ultrasound-guided fine needleaspiration (FNA) biopsy. Although FNA can identify the majority of nodules as either benignor malignant, one-third of aspirates demonstrate indeterminate cytologic characteristics.Though most of these nodules will be pathologically benign, thyroid surgery has usually beenneeded to make an accurate diagnosis, and the extent of surgery needed (lobectomy versustotal thyroidectomy) is difficult to predict in advance. New molecular techniques are beinginvestigated and used clinically to improve decision making in patients with thyroid noduleswith indeterminate cytology. These molecular markers have the potential to help cliniciansdecide which patients may be monitored without thyroid surgery, and also the optimalstrategy when surgery is felt to be necessary.

KEYWORDS: genetic markers • indeterminate thyroid cytology • molecular markers • mutation panel • thyroid cancer

• thyroid nodules

Nodular thyroid disease is common in the gen-eral population. Although palpation alone iden-tifies thyroid nodules in 3–7% of the patients,the prevalence of nodules detected by ultra-sound (US) ranges from 20–76% [1]. Withincreasing use of ultrasound, the discovery ofthyroid nodules has escalated. Since the risk ofmalignancy in incidentally detected thyroidnodules is only 3.5–5% [1], the clinical chal-lenge is to identify patients at high risk of devel-oping thyroid cancer and to avoid unnecessaryintervention in patients with low risk of malig-nancy. Several strategies have been proposed forrisk stratification in thyroid nodular diseaseincluding clinical history, serum thyrotropinand sonographic features. Ultrasound-guidedfine needle aspiration (FNA) has been the mostaccurate tool for identification of thyroid malig-nancy and can dramatically decrease the per-centage of patients with thyroid nodulesundergoing thyroid surgery [2].

FNA has substantially improved the charac-terization of thyroid nodules and the majority ofnodules can be characterized as benign or malig-nant. However, FNA has intrinsic limitationsand is indeterminate in ~25% of the cases [3].Most of these patients undergo thyroid surgery

for definitive pathologic diagnosis, though only6–30% of these excised lesions are determinedto be malignant, thus exposing a significantnumber of patients to unnecessary surgeries[4–7].The Bethesda classification system has furthercharacterized indeterminate cytology into threecategories: atypia of undetermined significance(AUS)/follicular lesion of undetermined signifi-cance (FLUS) with a malignancy risk of 5–15%;follicular or oncocytic neoplasm/suspicious forfollicular or oncocytic neoplasm (FN/SFN)with a malignancy risk of 15–30%; and suspi-cious for malignancy (SFM) with a malignancyrisk of 60–75% [3]. Despite the proposed tiersystem for indeterminate lesions, practical man-agement of these lesions remains controversialand diagnostic surgery is common. Tools toreduce these diagnostic surgeries for indetermi-nate thyroid nodules would be welcomed bypatients and physicians alike.

A further clinical challenge is to decide theextent of initial surgery in the setting of indeter-minate cytology. A patient who has a diagnosticlobectomy but is found pathologically to havethyroid cancer will generally be recommendedto have a completion thyroidectomy (unless thetumor is <1 cm). Conversely, a patient who has

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a total thyroidectomy, but turns out to have a benign lesion willhave undergone a more extensive, higher risk procedure thannecessary, and will also then require lifelong thyroid hormonereplacement [8,9]. Tools that would guide in the selection of theappropriate surgical procedure would also be welcomed. Severalmolecular techniques have been investigated in an effort to over-come the inherent limitations of indeterminate cytology and arereviewed below.

Immunohistochemical markersMany studies have employed immunohistochemical markers –including galectin-3, HBME-1, CK-19, TPO and DDP-4 – todistinguish between follicular-patterned lesions on FNA. Galec-tin is a type of lectin protein that binds with b-galactosides onthe cell surface. Galectin is involved in cell adhesion, cell cycleregulation, apoptosis and tumor progression. Well-differentiatedthyroid cancers often express galectin-3, whereas normal thyroidtissue and benign nodular proliferations generally do not. How-ever, studies of galactin-3 staining in indeterminate thyroidcytology specimens have shown a wide variation in results, withsensitivities ranging from 62 to 100% [10–13].

In one of the largest studies of galectin-3, Bartolazzi et al. ana-lyzed galectin-3 staining preoperatively on indeterminate thyroidnodule cytology and compared the results with the final histopa-thologic diagnosis [14]. Galectin-3 expression was detected in134 of 465 (29%) of indeterminate FNA samples, and of these,101 of 134 (75%) were found to be malignant. In the 331 of465 (71%) indeterminate FNA samples that did not expressgalectin-3, 280 (85%) were found to be benign on final histopa-thology diagnosis. The sensitivity of galectin for detection ofmalignancy was 78%, specificity was 93%, positive predictivevalue (PPV) was 82% and negative predictive value was 91%. Bythe use of the galectin-3 test, 88% of FNA samples were cor-rectly classified. The authors concluded that if galectin-3 wasincluded in surgical decisionmaking, then more than two-thirdsof unnecessary thyroid surgical procedures could be avoided.However, nearly 1 in 10 thyroid cancers would be missed.

To try to improve the detection of thyroid cancer,Prasad et al. tested galectin-3 with a panel of markers includingfibronectin-1, CITED-1, CK-19 and HBME-1 [15]. Galectin-3was found to be the most sensitive marker, whereas CK-19 andCITED-1 did not add any additional information. They con-cluded that the panel including galectin-3, fibronectin-1 andHBME-1 detected malignancies most efficiently, though with afalse-positive rate of 24% due to expression of these markerson follicular adenomas (FAs).

De Matos et al. evaluated 170 thyroid lesions by immuno-histochemical staining with HBME-1, galectin-3 and CK-19.HBME-1 was the most sensitive (84%) and specific (48%) markerfor detection of malignancy, whereas galectin-3 and CK-19 wereuseful in differentiating between follicular adenoma (FA),follicular-variant papillary thyroid cancer (FVPTC) and follicularthyroid carcinoma (FTC) [16].

Thyroid peroxidase (TPO) immunostaining is reduced inthyroid biopsy specimens from malignant transformation.

Yousaf et al. evaluated TPO immunostaing in core needle thy-roid biopsy specimens and reported that >80% staining wasassociated with benign histology (diagnostic sensitivity 89%and specificity 97%). Malignant thyroid nodules showed <80%staining with TPO immunostaining providing additional diag-nostic tool for differentiation of benign versus malignantlesions [17]. De Micco et al. compared the utility of HBME-1,anti-TPO and Dipeptidyl aminopeptidasetype 4 (DPP-4) stain-ing for diagnosis of malignancy in thyroid cytology specimens.TPO staining was most sensitive and DPP-4 staining was iden-tified to be most specific for the diagnosis of maliganancy [18].

Liquid-based cytology (LBC) techniques is increasingly beingutilized to improve diagnostic accuracy of indeterminate speci-mens [19]. Advantages of LBC include not only simplicity oftechnique for the physician performing fine needle aspirationbiopsy (FNAB) but also excellent cell preservation, lack of back-ground and availability of stored cellular material for furtherimmunohistochemical and molecular diagnostic studies.Fadda et al. applied an immuhistochemical panel (HBME-1 andGalectin-3) to indeterminate cytology specimens prepared inLBC and identified that the panel was positive in 83.3% ofmalignant and negative in 87.5% of benign histologic cases [20].In general, studies of immunohistochemical markers have dem-onstrated insufficient sensitivity and specificity for routine clini-cal use, and interest has shifted to other molecular techniques.

Somatic mutations & gene rearrangementsIn recent years, there has been significant advancement in theunderstanding of molecular pathways involved in thyroidcarcinogenesis. The MAPK and PI3/AKT pathway regulate cellproliferation, differentiation and apoptosis. Identification ofmutations and gene rearrangements associated with activationof MAPK as well as PI3/AKT signaling pathways show promiseas guides to targeted anticancer therapies for advanced thyroidcancer patients. Inhibitors of BRAF, RAS and PI3K add to thearmamentarium of available therapeutic options [21].

Genetic alterations identified in FNA specimens may alsohave diagnostic utility (TABLE 1).

BRAF

BRAF gene encodes a serine threonine protein kinase (B-Raf).Mutations of BRAF result in activation of MAPK and PI3K/AKT pathways. In particular, an activating mutation of BRAFcaused by substitution of glutamic acid for valine at position600 (V600E) is implicated in 51% of classic papillary carcino-mas and 24% of FVPTC [22]. BRAF activation is also associ-ated with K601E point mutation, small in-frame insertions ordeletions surrounding codon 600. BRAF mutation has alsobeen detected in anaplastic and poorly differentiated carcino-mas that develop from de-differentiation of PTC. Since BRAFV600 mutation is not typically seen in follicular carcinoma orbenign thyroid nodules, it is considered as a specific marker ofpapillary thyroid cancer [22].

BRAF is also associated with aggressive biologic behavior,extrathyroidal extension and tumor recurrence. In long-term

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studies, BRAF is associated with higher rate of recurrent/persistent disease and decreased survival. It has been suggestedthat patients with cytologic specimens positive for BRAFshould undergo total thyroidectomy with central compartmentlymphadenectomy [23,24].

RAS

RAS gene family is composed of three genes (HRAS, NRAS andKRAS). These genes encode small GTPase proteins (Ras pro-teins) that propagate signals arising from the cell membrane.Point mutations of RAS gene activates these G proteins leadingto activation of MAPK and PI3K/AKT pathways. Point muta-tions of RAS gene increase the affinity of RAS proteins for GTP(mutations in codons 12 and 13) or inactivate its autocatalyticGTPase function (mutations in codon 61). Mutations involvingNRAS and HRAS codon 61 are most commonly involved inthyroid cancer, though mutations have been found in all threegenes. Most of the RAS mutations identified in PTC are associ-ated with FVPTC (20–45%). Lee et al. reported increased accu-racy of detection of FVPTC in cytology specimens positive forRAS mutation. Prevalence of RAS mutations in FTC is 40–50%, with NRAS mutation in particular being associated withaggressive behavior of FTC [25]. RAS mutations are also found in20–40% of follicular adenoma. RAS-positive FAs have macrofol-licular and colloid-rich histology, and it has been hypothesizedthat these lesions represent carcinoma in situ [26].

PAX8/PPAR g rearrangement

PAX8/PPAR g rearrangement results from translocation t (2; 3)(q13; p25) leading to the fusion between PAX8 gene andPPARg gene. PAX8 gene codes for thyroid-specific paireddomain transcription factor. PPARg gene codes for peroxisomeproliferator receptor. PAX8/PPAR g rearrangement leads tooverexpression of PPAR g receptor and inhibits the antiproli-ferative activity of PPAR g receptor.

This rearrangement is identified in 20–40% of follicular car-cinomas, a small percentage of the oncocytic variant of FTCand occasionally in follicular variant of papillary carcinomas.PAX8/PPAR g rearrangement has been associated with solid

pattern of tumors, invasiveness and earlier age at presentation.PAX8/PPAR g rearrangement has also been detected in 2–10%FAs, which couldrepresent in situ carcinomas or follicular carci-nomas where invasion was not identified on histologicalanalysis [26].

RET/PTC rearrangement

RET proto-oncogene encodes a receptor tyrosine kinase. Acti-vation of tyrosine kinase receptor is implicated in the activationof MAPK and PI3 pathways. Intrachromosomal rearrangementsRET/PTC1 and RET/PTC3 produce constitutively activatedform of this signaling complex. RET/PTC rearrangementsresult from fusion of 3´-RET receptor tyrosine kinase gene and5´-portion of various other genes.

RET/PTC rearrangements are seen in only 20% of adultsporadic PTC, but are more frequent in patients with a history ofradiation exposure (50–80%) and in PTC of children and youngadults (40–70%) [26]. For example, RET/PTC3 rearrangement isthe most common mutation in children with PTC within 10 yearsof radiation exposure from Chernobyl [26]. RET/PTC positivePTC presents at a younger age and is more likely to have lymphnode metastases at the time of initial diagnosis. RET/PTC rear-rangements are also detected in FAs, thus limiting the utility ofthis marker in the diagnostic evaluation of thyroid nodules [26].

Combined genetic analysisMutations in BRAF, RAS and RET/PTC are found in 70% ofPTC and may be associated with aggressive behavior [26]. Fol-licular carcinomas (80%) harbor RAS or PAX8/PPAR g muta-tions [26]. Nikiforova et al. evaluated the diagnostic utility of apanel of mutations on routine FNA samples [26]. During FNA of470 nodules, additional material was collected for analysis ofpoint mutations and gene rearrangements. DNA was tested forBRAF V600 E, K601E, NRAS codon 61, HRAS codon 61 andKRAS codons 12 and 13 point mutations using real-time lightcycler PCR and fluorescence melting curve analysis. Gene rear-rangements for RET/PTC 1, RET/PTC 2, RET/PTC 3 andPAX8/PPARg were detected by RT-PCR from RNA. Moleculartesting identified 32 mutations in 462 FNA samples: 18BRAFV600 E, 5NRAS codon 61, 3HRAS codon 61, 4RET/PTC1,1RET/PTC 3 and 1PAX8/PPARg . After surgery, 31(97%) muta-tion positive nodules were positive for malignancy. Sensitivity ofmutation analysis for identification of malignancy was 62%. Allnodules with indeterminate cytology (52 cytology specimens),which were positive for mutation analysis were also malignant bysurgical pathology. BRAF was the most common mutation andhad a 100% positive predictive value (PPV) for PTC (TABLE 2).RAS was the second most common mutation and had an 87.5%positive predictive value for malignancy. One of the eight RASpositive nodules was a FA.

Another prospective study by Nikiforova et al. evaluated therole of mutation panel analysis (BRAFV600 E, NRAS codon61, HRAS codon 61, KRAS codons 12/13 point mutationsand RET/PTC 1, RET/PTC 2, RET/PTC 3 and PAX8/PPARg) in a large cohort of indeterminate specimens [22]. In

Table 1. Mutational profile of differentiatedthyroid cancer.

Types of DTC Prevalence of mutations

PTC BRAF: 40–45%

RAS: 10–20%

RET/PTC: 10–20%

TRK: <5%

Follicular thyroid carcinoma RAS: 40–45%

PAX8/PPARg: 30–35%PIK3CA:<10%

PTEN: <10%

Oncocytic variant of follicular

thyroid carcinoma

NDUFA13: 10–20

PTC: Papillary thyroid cancer.

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indeterminate categories (atypia of undetermined significance[AUS], follicular lymohoma [FN; including Hurthle cell var-iant] and SFM), identification of positive mutation panel isassociated with increased risk of malignancy (PPV of 88,87 and 95%, respectively). It has been suggested that becauseof the high risk of malignancy, patients with indeterminatecytology who are mutation positive should be considered ascandidates for total thyroidectomy [23] (TABLE 3). Utilization ofLBC technique for mutational panel analysis could improve thesensitivity of mutation detection in indeterminate cytologyspecimens [27]. Next-generation sequencing (NGS) allowssequencing of large regions of genome with increased detectionrate of various genetic variants. Identification of low prevalencemutations (PI3KCA, AKT1, PTEN, TP53 genes and chromo-somal rearrangements of the BRAF and NTRKI genes) withNGS is likey to improve the accuracy of current mutationalpanel [28]. Furthermore, utilization of LBC technique for muta-tional panel analysis could improve sensitivity of mutationdetection in indeterminate cytology specimens [27].

Preoperative testing for mutation panel not only may behelpful diagnostically but also could help provide prognosticinsight. Mutation negative cancers had lower risk of inva-siveness and extrathyroidal extension. BRAF mutation inPTC is associated with aggressiveness of disease and increaserate of recurrence. Central neck dissection has been pro-posed to reduce the rate of recurrence in high-risk BRAF-positive PTC [29].

Cost–effectiveness of a molecular testing panel was evaluatedby Yip et al. in a hypothetical group of patients using a deci-sion tree model [30]. Molecular testing added $104 per patientto the cost of thyroid nodule evaluation. Numbers of lobecto-mies were reduced with the addition of molecular testing andnumbers of total thyroidectomies were increased. Molecular test-ing added a cost of $5,031 to the cost of each total thyroidec-tomy ($11,383). The overall cost of performing lobectomy($7,684) followed by completion thyroidectomy ($11,954) wasstill higher by comparison. In a sensitivity analysis, savings weredemonstrated when molecular testing cost was less than $870. Bycontrast, a commercially available panel in the USA (Asuragen’smiRInform panel) is available at a cost of $2250 [31].

Methylation of tumor suppressor genes is known to result inaberrant activation of PI3K/Akt and BRAF/MEK/MAPK path-ways. Thyroid cancer subtypes have been determined by DNAarrays to have differential methylation pattern. Tumor-specificDNA methylation pattern could potentially identify thyroid can-cer subtypes in thyroid cytology. Hypermethylation pattern atgene promoters in thyroid cancer subtypes might provide poten-tial therapeutic options with demethylation agents [32].

Genetic expression classifierMicroarray technology identifies patterns of expressed mRNAin biologic specimens. In thyroid cytology, the goal has been todevelop a gene expression algorithm that can reliably indicatewhether a thyroid nodule is benign or malignant.

The genetic expression classifier (GEC) called Afirma analyzesthe expression of 167 RNA transcripts from thyroid FNA speci-mens and shows promise in improving preoperative risk assess-ment in indeterminate nodules. An initial validation reported anegative predictive value of 96% for identifying benign nodulesin a sample of indeterminate specimens [4]. These results werevalidated in a large, prospective, 19 months multicenter trialenrolling 3789 patients [33]. Over 413 out of 577 indeterminateFNAB specimens had histopathologic diagnosis from surgicalspecimens as reference standard. There were 265 indeterminatenodules included in the analysis: 49% of the samples wereFLUS, 31% FN/SFN and 21% were SFM. Only 85/265 aspi-rates (32%) were classified as malignant on blinded histopatho-logical review. Of these 85 cancers, 78 samples were identified as‘suspicious’ by GEC, whereas seven samples were false negative.False-negative results were noticed in smaller nodules (1.3 cmversus 2.2 cm), and 6 of 7 false-negative specimens had a lowerfollicular cell content. Percentages of malignant lesions in thethree categories were: FLUS (24%), FN/SFN (25%), SFM(62%), yielding negative predictive value of 95, 94 and 85%,

Table 2. Performance of point mutations andchromosomal rearrangement analysis according tothe final histopathologic diagnosis forindeterminate samples.

n Malignant Benign Performanceof molecularanalysis panel

Follicular

lesion of

undetermined

significance

21 Sensitivity: 100%

Specificity:100%

PPV: 100%

NPV: 100%

Mutation

positive

3 3 0

Mutation

negative

18 0 18

Follicular

neoplasm/

Hurthle

cell neoplasm

23 Sensitivity: 75%

Specificity: 100%

PPV: 100%

NPV: 79%

Mutation

positive

9 9 0

Mutation

negative

18 3 11

Suspicious for

malignancy

7 Sensitivity: 60%

Specificity: 100%

PPV: 100%

NPV: 50%

Mutation

positive

3 3 0

Mutation

negative

4 2 2

NPV: Negative predictive value; PPV: Positive predictive value.Data taken from [26].

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respectively (TABLE 5). It has been proposed that the Afirma test,with its high negative predictive value (NPV), be used as a ‘ruleout’ test to reduce the number of diagnostic surgeries for patientwith FLUS or FN/SFN cytologies (the NPV for SFM is nothigh enough to recommend deferring surgery for these patients).Unfortunately, the positive predictive value of Afirma is nothigh, so many patients with ‘suspicious’ GEC results will still bedetermined to have benign lesions after surgery.

The cost–effectiveness of GEC was assessed in a study byLi et al. in which decision analysis of a hypothetical group ofpatients was conducted using GEC [34]. The cost of GEC wasestimated to be $3,200. Using current guidelines for evaluationand management of thyroid nodules, they reported that 74% ofunnecessary surgeries were avoided with the use of GEC, and perpatient cost of care could be reduced from $12,172 to $10,719.

Thyrotropin receptor mRNAThyrotropin receptor (TSHR) mRNA is an additional clinicaltool for evaluation of patients with thyroid nodules.Milas et al. evaluated TSHR mRNA as a molecular marker ofthyroid cancer [35]. TSHR mRNA is detected by RT-PCR ofRNA extracted from mononuclear cell fraction of a blood sam-ple utilizing TSHR mRNA primers. TSHR mRNA is a molec-ular marker for circulating thyroid cancer cells. Levels of this

molecular marker have been found to be increased in patientswith thyroid cancer, and TSHR mRNA has been studied as adiagnostic marker to determine the risk of cancer in patientswith indeterminate cytology [36,37].

In a prospective study by Milas et al., 54 patients with thy-roid cytology indicating FN underwent lobectomy followed bycompletion thyroidectomy if surgical pathology was consistentwith malignancy [38]. Positive TSHR mRNA was able to iden-tify 22 of 29 patients with malignancy. When suspicious USfeatures were used as adjunct to TSHR mRNA to predictmalignancy, there were 28 of 29 patients who were correctly

Table 3. Proposed clinical management of thyroid nodules with integrated approach of cytology, pointmutations and chromosomal analysis.

Cytologic diagnosis Cancer riskbased on

cytology (%)

Mutationalstatus

Cancer risk basedon cytology and

molecular analysis

Goal of molecularanalysis

Proposedclinicalmanagement

Nondiagnostic Avoid the need of

second FNAB

Benign 2.1 Mutation

negative

0.9 Reduce false-negative

rate

Clinical and

radiologic

follow-up

AUS/FLUS 14 Mutation

positive

88 Reduce diagnostic

thyroid surgeries

Total

thyroidectomy

Mutation

negative

5.9 Repeat FNA ±

observation

Follicular neoplasm/

suspicious for follicular or

oncocytic neoplasm

27 Mutation

positive

87 Reduce rate of

completion

thyroidectomies

Total

thyroidectomy

Mutation

negative

14 Lobectomy

SFM 54 Mutation

positive

95 Increase rate of total

thyroidectomy

Total

thyroidectomy

Mutation

negative

28 Lobectomy

Malignant 100 Mutation

positive

100 Define the extent of

initial surgery

Total

thyroidectomy

+ CND

AUS: Atypia of undetermined significance; CND: Central neck dissection; FNA: Fine needle aspiration; FNAB: Fine needle aspiration biopsy; SFM: Suspicious formalignancy.

Table 4. Thyrotropin receptor mRNA topredict malignancy in thyroid cytology indicatingfollicular neoplasm.

Malignant Benign TSHRmRNAperformance

n = 54 Sensitivity: 76%

TSHRmRNA positive 22 1 Specificity: 96%

TSHRmRNA negative 7 24 PPV: 96%

NPV: 77%

TSHR: Thyrotropin receptor.Data taken from [35].

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predicted to have thyroid cancer. The addition of suspiciousUS features (hypervascularity, microcalcifications, irregularshape, indistinct margins, solid and hypoechoic nodule) to analgorithm using TSHR mRNA algorithm increases the sensitiv-ity of detection of malignancy to 97%, specificity to 84%, posi-tive predictive value to 88% and negative predictive value to95% (TABLE 4). According to an algorithm suggested by Milas et al.[35] initial thyroidectomy rather than lobectomy should be con-sidered if FNAB is concerning for FN or indeterminate andTSHR mRNA ‡1 ng/mg. Risk of thyroid cancer in this situationis 90–96%. There is a degree of overlap between benign nodulesand thyroid cancer at TSHR mRNA levels 1–1.5 ng/mg. TSHRmRNA >5 ng/mg was strongly associated with thyroid cancer.This test is available only through the Cleveland Clinic labora-tory, and all currently available data are from this institution [31].

miRNA expressionmiRNAs are small (18–24 nt) noncoding RNAs that bind to3´-untranslated regions of target mRNA and inhibit proteinsynthesis. It has been reported that miRNAs control cell proc-esses by regulating gene expression at posttranscriptional leveland affecting cell proliferation and apoptosis [39]. More than10,000 messenger RNAs are regulated by miRNAs. miRNAsmay be overexpressed or downregulated in malignant cells, andspecific tumors exhibit differential expression profile of miR-NAs (TABLE 6). Expression profiles of miRNAs may differentiatebetween benign versus malignant lesions. These differences in

expression profile have been investigated for diagnostic purposesin thyroid nodules. miRNA molecules are relatively stable incytology specimens and fresh frozen samples that make themavailable for use as diagnostic marker.

In a study by Weber et al., miRNA-197 and miRNA-346were overexpressed in FTC as compared with FAs [40].Sheu et al., reported that miRNAs (miRNA-146b, -181b, -21, -221, -222) are overexpressed in a follicular variant of PTC andFTC as compared to FA and multinodular goiter [41]. There is astrong correlation between the miRNA expression and somaticmutation status. miRNA-187 was expressed at high level in PTCwith RET/PTC rearrangement. BRAF and RAS positive tumorsand tumors with no mutations have high expression of miRNA-221 and miRNA-222. PTC with RAS mutation had thehighest expression profile of miRNA-146b. Shen et al., evaluatedfrozen PTC samples and reported that miRNA-146b, -221and-222) are upregulated in PTC [42].

In another study, the same group evaluated the diagnosticutility of miRNA in samples. Expression levels of four miR-NAs-146b, -221, -187, -30d could differentiate between benignand malignant nodules. For PTC samples, these four miRNAshave 95.8% positive predictive value for malignancy. Positivepredictive value for FTC cases was lower at 69.2%. When eval-uating nodules, cytology reads as FLUS, these four miRNAs-146b,-221, -187, -30d can detect PTC cases but may be mis-leading in FTC and FVPTC. Kitano et al., evaluated theexpression level of four miRNA (miR-7,-126, 374a, let-7g) in95 FNA specimens [43]. According to their predictor model,miR-7 had the highest negative predictive value. In a subgroupanalysis of nondiagnostic, indeterminate and suspicious sam-ples, miR-7 had a negative predictive value of 100%.

Keutgen et al. evaluated the expression profiles of miRNAon indeterminate specimens and determined their prognosticvalue on final histologic diagnosis [44].

Expression of miRNAs (miRNA-222, miRNA-328,miRNA-197, miRNA-21) combined in a predictive model accu-rately differentiates benign from malignant lesions. After exclud-ing Hurthle cell neoplasm, accuracy improved to 97% withsensitivity of 100% for detection of malignancy. Hurthle cell

Table 5. Performance of genetic expression classifier in indeterminate cytology specimens.

Indeterminatenodules

n (%) Prevalence ofmalignantlesions (%)

Sensitivity (%) Specificity (%) Positivepredictivevalue (%)

Negativepredictivevalue (%)

Total 265 32 92 52 47 93

Follicular lesion of

undetermined

significance

129 (49.0) 24 90 53 38 95

Follicular neoplasm/

suspicious for follicular

neoplasm

81 (30.6) 25 90 49 37 94

Suspicious for

malignancy

55 (20.8) 62 94 52 76 85

Data taken from [33].

Table 6. miRNA profile of thyroid cancer.

Type of thyroid cancer miRNA profile

PTC miR-146b, 221, 222

FTC miR-197, 346, 155, 224

Anaplastic cancer miR-30d, 125b, 26a, 30a-5p

Follicular-variant PTC miR 375, 551-b, 181-2-3p, 99b-3p

Oncytic variant of FTC miR-885-5p

FTC: Follicular thyroid carcinoma; PTC: Papillary thyroid cancer.

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neoplasms represent a different miRNA expression profile andseparate prediction model utilizing different miRNA should beused to evaluate for Hurthle adenomas versus carcinomas.

Conventional and oncocytic FTCs show upregulation ofmiRNA-885-5p [45]. Use of these miRNA could potentially beuseful in follicular pattern lesions. FVPTC has been identifiedto have unique expression profile of miRNA. miRNA dysregu-lated in FVPTC include miRNA 375, 551-b, 181-2-3p and99b-3p. miRNAs 181-2-3p and 99b-3p were associated withadverse clinical outcome [46].

Despite the potential utility of miRNA in thyroid noduleevaluation, diagnostic use of miRNA has not been extensivelyevaluated in large multicenter trials, and its use is currentlyinvestigational.

Expert commentary & 5-year viewThyroid cytology from FNA remains the standard for evaluat-ing thyroid nodules. Patients with benign cytology may gener-ally be monitored expectantly, whereas patients with malignantcytology should have thyroid surgery. Molecular markers donot replace conventional FNA cytology but instead provide acomplementary tool particularly in the evaluation of indetermi-nate lesions. Ideally, a molecular marker should be simple touse, widely available, reproducible, cost–effective and reliablydifferentiate benign from malignant nodules [47]. None of themethodologies currently available or under investigation meetall these criteria, and each has advantages and disadvantages.To date, immunohistochemical markers lack the sensitivity orspecificity to guide treatment, whereas studies of TSHRmRNA have been limited to one institution. miRNA testingappears promising, but remains investigational. The mostrobust data exist for GEC and mutation analysis.

GEC has a high negative predictive value and has beenpromoted as a ‘rule out’ test for patients with indeterminatecytology (i.e., patients with indeterminate cytology and abenign GEC profile are unlikely to harbor malignancy andmight be safely monitored without surgical intervention).This seems a reasonable approach for patients with FLUS orFN/SFN cytologies, but the high malignancy rate (and lowerGEC negative predictive value) in SFM patients would arguefor thyroid surgery in SFM patients regardless of GEC sta-tus. It has also been argued that by simply repeating theFNA in patients with FLUS cytology reveals a definitivediagnosis in >50% of patients [3,48]. The performance ofGEC for patients with cytology suggesting Hurthle celllesions seems similar to other follicular lesions, though thenumber of patients studied has been limited. At present,GEC should be considered on an individual basis forpatients with FNA specimens demonstrating FLUS or FN/SFN who would be comfortable deferring surgery in thepresence of a negative GEC.

Testing for somatic mutations has a high positive predictivevalue but a low sensitivity and has been promoted as a ‘rule in’test to determine the appropriate surgical approach for patientswith indeterminate cytology (i.e., patients with indeterminate

cytology and negative mutational analysis should undergo diag-nostic lobectomy, whereas patients with indeterminate cytologyand positive mutational analysis should undergo total thyroidec-tomy). Central neck dissection could be offered in BRAF-positivePTC. Long-term follow-up of this cohort of patients will behelpful to identify benefits on reducing the rate of recurrence andmortality. At present, mutational analysis should be consideredfor patients with indeterminate lesions who desire surgery butwho seek guidance on surgical extent.

In fact, a two-step methodology could be proposed forpatients with FLUS or FN/SFN cytologies. GEC would be uti-lized initially, with surgery deferred and a monitoring plan ini-tiated if the GEC profile was benign. If the GEC wassuspicious, mutational analysis would be performed. GEC sus-picious, mutation-negative patients would undergo lobectomy,whereas GEC suspicious, mutation-positive patients wouldundergo a total thyroidectomy as the initial surgery. The goalof a two-step approach would be to avoid any surgery in low-risk patients, to offer total thyroidectomy to higher riskpatients, and to leave very few patients in the position of need-ing a diagnostic lobectomy, which in retrospect will turn out tobe either too much treatment (in the case of a pathologicallybenign lesion) or too little treatment (in the case of a malig-nant lesion) (FIGURE 1). At present, such an approach would beprohibitively expensive. Large-scale prospective studies mayhelp to further refine the proposed algorithm. Evaluation withFNA remains the gold standard for assessment of thyroidnodules.

Future studies for molecular markers should include largenumbers of patients with indeterminate cytology. Standar-dized terminology for cytology as suggested in consensusguidelines should be used, so results are widely applicable inclinical practice. Novel diagnostic tools on the horizon willcontinue to add and refine management of indeterminate

Indeterminate cytology

SFM

Mutation panel analysis

FN/SFN

GEC or lobectomy

+ GEC– +

Potential role formiRNA

Total thyroidectomy,± central neck dissection

AUS/FLUS

Repeat FNA,or GEC,

or clinical follow upwith US

Figure 1. Proposed algorithm for the management ofindeterminate thyroid cytology.AUS: Atypia of undetermined significance; FLUS: Follicular lesionof undetermined significance; FN: Follicular neoplasm; FNA: Fineneedle aspiration; GEC: Genetic expression classifier; SFM:Suspicious for malignancy; SFN: Suspicious for follicular oroncocytic neoplasm; US: Ultrasound.

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thyroid cytology. Preoperative characterization of thyroidnodules requires a multidisciplinary approach of eachpatient. When indeterminate thyroid lesions can reliably bedistinguished preoperatively, a significant number ofunnecessary surgical procedures can be avoided, with thepotential to reduce both morbidity and associated healthcarecosts.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with

any organization or entity with a financial interest in or financial conflict

with the subject matter or materials discussed in the manuscript. This

includes employment, consultancies, honoraria, stock ownership or options,

expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• The Incidence of nodular thyroid disease and thyroidcancer has been increasing over the past two decades due to the increasing use of

high-frequency neck US. Mortality from thyroid cancer continues to be low (survival rate of nearly 100% for localized disease) [49]. Risk

stratification of thyroid nodules is of paramount importance to identify those patients who are most likely to benefit from intervention.

Management of patient with indeterminate nodules remains problematic. Using cytology and clinical factors alone, most patients will be

recommended for surgery, even though only a minority will have thyroid cancers on final pathology. Molecular markers are being inten-

sively investigated to refine management of these patients.

• Currently, commercially available molecular markers for thyroid cytology include Afirma, TSHR mRNA and miRInform mutation panel.

• Mutation analysis panel (BRAFV600 E, NRAS codon 61, HRAS codon 61, KRAS codons 12/13 point mutations and RET/PTC 1, RET/PTC

2, RET/PTC 3 and PAX8/PPAR g) shows a very high positive predictive value for thyroid cancer, but the sensitivity is low. These tests can

be used to guide surgical extent, but cannot be used to exclude malignancy.

• Genetic expression analysis identifies nodules that are likely to be benign with a high negative predictive value. This test has the

potential to reduce the number of unnecessary surgical procedure in patients with indeterminate cytology.

References

Papers of special note have been highlighted as:

• of interest

•• of considerable interest

1 Gharib H, Papini E, Paschke R et alAmerican association of clinical

endocrinologists, associazione medici

endocrinologi, and european thyroid

association medical guidelines for clinical

practice for the diagnosis and management

of thyroid nodules: executive summary of

recommendations. Endocr. Pract. 16(3),468–475 (2010).

2 Carpi A, Ferrari E, Toni MG, Sagripanti A,

Nicolini A, Di Coscio G. Needle aspiration

techniques in preoperative selection of

patients with thyroid nodules: a long-term

study. J. Clin. Oncol. 14(5), 1704–1712(1996).

3 Cibas ES, Ali SZ. The bethesda system for

reporting thyroid cytopathology. Thyroid19(11), 1159–1165 (2009).

4 Chudova D, Wilde JI, Wang ET et al.Molecular classification of thyroid nodules

using high-dimensionality genomic data.

J. Clin. Endocrinol. Metab. 95(12),5296–5304 (2010).

5 Nayar R, Ivanovic M. The indeterminate

thyroid fine-needle aspiration: experience

from an academic center using terminology

similar to that proposed in the

2007 National Cancer Institute thyroid fine

needle aspiration state of the science

conference. Cancer 117(3), 195–202 (2009).

6 Ohori NP, Nikiforova MN, Schoedel KE

et al. Contribution of molecular testing to

thyroid fine-needle aspiration cytology of

‘follicular lesion of undetermined

significance/atypia of undetermined

significance’. Cancer Cytopathol. 118(1),17–23 (2010).

•• Demonstrates the utility of molecular

analysis of cytologic specimens to

improve diagnostic utility.

7 Layfield LJ, Morton MJ, Cramer HM,

Hirschowitz S. Implications of the proposed

thyroid fine-needle aspiration category of

“follicular lesion of undetermined

significance”: a five-year multi-institutional

analysis. Diagn. Cytopathol. 37(10),710–714 (2009).

8 Duick DS. Overview of molecular

biomarkers for enhancing the management

of cytologically indeterminate thyroid

nodules and thyroid cancer. Endocr. Pract.18(4), 611–615 (2012).

9 Kim MI, Alexander EK. Diagnostic use of

molecular markers in the evaluation of

thyroid nodules. Endocr. Pract. 18(5),796–802 (2012).

10 Saggiorato E, De Pompa R, Volante M

et al. Characterization of thyroid ‘follicularneoplasms’ in fine-needle aspiration

cytological specimens using a panel of

immunohistochemical markers: a proposal

for clinical application. Endocr. Relat. Cancer12(2), 305–317 (2005).

11 de Matos LL, Del Giglio AB,

Matsubayashi CO, de Lima Farah M,

Del Giglio A, da Silva Pinhal MA.

Expression of CK-19, galectin-3 and

HBME-1 in the differentiation of thyroid

lesions: systematic review and diagnostic

meta-analysis. Diagn. Pathol. 7, 97 (2012).

12 Paunovic I, Isic T, Havelka M, Tatic S,

Cvejic D, Savin S. Combined

immunohistochemistry for thyroid

peroxidase, galectin-3, CK19 and

HBME-1 in differential diagnosis of thyroid

tumors. Apmis 120(5), 368–379 (2012).

13 Chiu CG, Strugnell SS, Griffith OL et al.Diagnostic utility of galectin-3 in thyroid

cancer. Am. J. Pathol. 176(5), 2067–2081(2010).

14 Bartolazzi A, Orlandi F, Saggiorato E et al.Galectin-3-expression analysis in the surgical

selection of follicular thyroid nodules with

indeterminate fine-needle aspiration

cytology: a prospective multicentre study.

Lancet Oncol. 9(6), 543–549 (2008).

15 Prasad ML, Pellegata NS, Huang Y,

Nagaraja HN, de la Chapelle A, Kloos RT.

Galectin-3, fibronectin-1, CITED-1,

HBME1 and

cytokeratin-19 immunohistochemistry is

useful for the differential diagnosis of

Review Khan & Laufgraben

446 Expert Rev. Endocrinol. Metab. 8(5), (2013)

Exp

ert R

evie

w o

f E

ndoc

rino

logy

& M

etab

olis

m D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 11

/03/

14Fo

r pe

rson

al u

se o

nly.

thyroid tumors. Mod. Pathol. 18(1), 48–57(2005).

16 de Matos PS, Ferreira AP,

de Oliveira Facuri F, Assumpcao LV,

Metze K, Ward LS. Usefulness of HBME-1,

cytokeratin 19 and galectin-3 immunostaining

in the diagnosis of thyroid malignancy.

Histopathology 47(4), 391–401 (2005).

17 Yousaf U, Christensen LH, Rasmussen AK

et al. Immunohistochemical staining for

thyroid peroxidase (TPO) of needle core

biopsies in the diagnosis of scintigraphically

cold thyroid nodules. Clin. Endocrinol.68(6), 996–1001 (2008).

18 de Micco C, Savchenko V, Giorgi R,

Sebag F, Henry JF. Utility of malignancy

markers in fine-needle aspiration cytology of

thyroid nodules: comparison of Hector

Battifora mesothelial antigen-1, thyroid

peroxidase and dipeptidyl aminopeptidase

IV. Br. J. Cancer 98(4), 818–823 (2008).

19 Cochand-Priollet B, Prat JJ, Polivka M et al.Thyroid fine needle aspiration: the

morphological features on thinprep slide

preparations. Eighty cases with histological

control. Cytopathology 14(6), 343–349 (2003).

20 Fadda G, Rossi ED. Liquid-based cytology in

fine-needle aspiration biopsies of the thyroid

gland. Acta Cytol. 55(5), 389–400 (2011).

21 Haugen BR, Sherman SI. Evolving

approaches to patients with advanced

differentiated thyroid cancer. Endocr. Rev.34(3), 439–455 (2013).

•• Comprehensive review of newer targeted

therapies for advanced thyroid cancer.

22 Nikiforov YE, Nikiforova MN. Molecular

genetics and diagnosis of thyroid cancer.

Nat. Rev. Endocrinol. 7(10), 569–580(2011).

23 Yip L, Nikiforova MN, Carty SE et al.Optimizing surgical treatment of papillary

thyroid carcinoma associated with BRAF

mutation. Surgery 146(6), 1215–1223 (2009).

24 O’Neill CJ, Bullock M, Chou A et al.BRAF(V600E) mutation is associated with

an increased risk of nodal recurrence

requiring reoperative surgery in patients with

papillary thyroid cancer. Surgery 148(6),1139–1145, discussion 1145–1136 (2010).

25 Fukahori M, Yoshida A, Hayashi H et al.The associations between RAS mutations

and clinical characteristics in follicular

thyroid tumors: new insights from a single

center and a large patient cohort. Thyroid22(7), 683–689 (2012).

•• Analyses the significance of RAS

mutations in follicular adenoma and

follicular carcinoma.

26 Nikiforov YE, Steward DL,

Robinson-Smith TM et al. Molecular

testing for mutations in improving the

fine-needle aspiration diagnosis of thyroid

nodules. J. Clin. Endocrinol. metab. 94(6),2092–2098 (2009).

27 Rossi ED, Martini M, Capodimonti S et al.Diagnostic and prognostic value of

immunocytochemistry and BRAF mutation

analysis on liquid-based biopsies of thyroid

neoplasms suspicious for carcinoma. Eur. J.Endocrinol. 168(6), 853–859 (2013).

28 Nikiforov YE, Yip L, Nikiforova MN. New

strategies in diagnosing cancer in thyroid

nodules: impact of molecular markers. Clin.Cancer. Res. 19(9), 2283–2288 (2013).

29 Alzahrani AS, Xing M. Impact of lymph

node metastases identified on central neck

dissection (CND) on the recurrence of

papillary thyroid cancer: potential role of

BRAFV600E mutation in defining

CND. Endocr.Relat. Cancer 20(1), 13–22(2013).

30 Yip L, Farris C, Kabaker AS et al. Costimpact of molecular testing for

indeterminate thyroid nodule fine-needle

aspiration biopsies. J. Clin. Endocrinol.Metab. 97(6), 1905–1912 (2012).

•• Evaluates the clinical implication of

BRAF +cytology specimens.

31 Hodak SP, Rosenthal For The American

Thyroid Association Clinical Affairs

Committee DS. Information for clinicians:

commercially available molecular diagnosis

testing in the evaluation of thyroid nodule

fine-needle aspiration specimens. Thyroid23(2), 131–134 (2013).

•• Brief discussion of commercially available

molecular markers in USA.

32 Rodriguez-Rodero S, Fernandez AF,

Fernandez-Morera JL et al.DNA methylation signatures identify

biologically distinct thyroid cancer subtypes.

J. Clin. Endocrinol. Metab. 98(7),2811–2821 (2013).

33 Alexander EK, Kennedy GC, Baloch ZW

et al. Preoperative diagnosis of benignthyroid nodules with indeterminate cytology.

N. Engl. J. Med. 367(8), 705–715 (2012).

•• Large, prospective multicenter study

validating GEC in indeterminate cytology

specimen.

34 Li H, Robinson KA, Anton B, Saldanha IJ,

Ladenson PW. Cost-effectiveness of a novel

molecular test for cytologically

indeterminate thyroid nodules. J. Clin.Endocrinol. Metab. 96(11), e1719–e1726(2011).

35 Milas M, Mazzaglia P, Chia SY et al. Theutility of peripheral thyrotropin mRNA in

the diagnosis of follicular neoplasms and

surveillance of thyroid cancers. Surgery141(2), 137–146, discussion 146 (2007).

36 Chia SY, Milas M, Reddy SK et al.Thyroid-stimulating hormone receptor

messenger ribonucleic acid measurement in

blood as a marker for circulating thyroid

cancer cells and its role in the preoperative

diagnosis of thyroid cancer. J. Clin.Endocrinol. Metab. 92(2), 468–475 (2007).

37 Wagner K, Arciaga R, Siperstein A et al.Thyrotropin receptor/thyroglobulin

messenger ribonucleic acid in peripheral

blood and fine-needle aspiration cytology:

diagnostic synergy for detecting thyroid

cancer. J. Clin. Endocrinol. Metab. 90(4),1921–1924 (2005).

38 Milas M, Shin J, Gupta M et al.Circulating thyrotropin receptor mRNA as a

novel marker of thyroid cancer: clinical

applications learned from 1758 samples.

Ann. Surg. 252(4), 643–651 (2010).

39 Gomez Saez JM. Diagnostic and prognostic

markers in differentiated thyroid cancer.

Curr. Genomics 12(8), 597–608 (2011).

•• Discusses the current understanding of

molecular and epigenetic markers in

thyroid tumorigenesis.

40 Weber F, Teresi RE, Broelsch CE,

Frilling A, Eng C. A limited set of human

microRNA is deregulated in follicular

thyroid carcinoma. J. Clin. Endocrinol.Metab. 91(9), 3584–3591 (2006).

41 Sheu SY, Grabellus F, Schwertheim S,

Worm K, Broecker-Preuss M, Schmid KW.

Differential miRNA expression profiles in

variants of papillary thyroid carcinoma and

encapsulated follicular thyroid tumours. Br.J. Cancer 102(2), 376–382 (2010).

42 Shen R, Liyanarachchi S, Li W et al.MicroRNA signature in thyroid fine needle

aspiration cytology applied to “atypia ofundetermined significance” cases. Thyroid22(1), 9–16 (2012).

43 Kitano M, Rahbari R, Patterson EE et al.Evaluation of candidate diagnostic

microRNAs in thyroid fine-needle aspiration

biopsy samples. Thyroid 22(3), 285–291

(2012).

44 Keutgen XM, Filicori F, Crowley MJ et al.A panel of four miRNAs accurately

differentiates malignant from benign

indeterminate thyroid lesions on fine needle

aspiration. Clin. Cancer Res. 18(7),2032–2038 (2012).

45 Dettmer M, Vogetseder A, Durso MB et al.MicroRNA expression array identifies novel

Diagnostic and prognostic implications Review

www.expert-reviews.com 447

Exp

ert R

evie

w o

f E

ndoc

rino

logy

& M

etab

olis

m D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 11

/03/

14Fo

r pe

rson

al u

se o

nly.

diagnostic markers for conventional and

oncocytic follicular thyroid carcinomas.

J. Clin. Endocrinol. Metab. 98(1), e1–e7(2013).

46 Dettmer MS, Perren A, Komminoth P,

Moch H, Nikiforov YE, Nikiforova MN.

Micro-RNA expression profiles in follicular

variant of papillary thyroid carcinomas.

Thyroid. 22 (Suppl. 1), (2012).

47 Kouniavsky G, Zeiger MA. The quest for

diagnostic molecular markers for thyroid

nodules with indeterminate or suspicious

cytology. J. Surg. Oncol. 105(5), 438–443(2012).

48 Faquin WC. Can a gene-expression classifier

with high negative predictive value solve the

indeterminate thyroid fine-needle aspiration

dilemma? Cancer cytopathol. 121(3),116–119 (2013).

•• Discusses high negative predictive value

of GEC in comparison with rebiopsy.

49 American Cancer Society. Cancer Facts &

Figures 2012. American Cancer Society,

Atlanta, GA, USA (2012).

Review Khan & Laufgraben

448 Expert Rev. Endocrinol. Metab. 8(5), (2013)

Exp

ert R

evie

w o

f E

ndoc

rino

logy

& M

etab

olis

m D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 11

/03/

14Fo

r pe

rson

al u

se o

nly.