Immunohistochemical and Molecular Markers ... - KoreaMed Synapse

R EV IEW ARTICLE

ISSN: 2005-162X

Clin Exp Thyroidol 2015 May 8(1): 50-60 http://dx.doi.org/10.11106/cet.2015.8.1.50

Immunohistochemical and Molecular Markers Associated with Differentiated Thyroid Carcinoma Jun Woo Jung1, June Young Choi1, Kyu Eun Lee2 and Kwi Won Park2 Department of Surgery, Seoul National University Bundang Hospital, Seongnam1, Department of Surgery, Seoul National University Hospital, Seoul2, Korea In the last decade, conventional diagnosis of thyroid nodules largely depended on fine-needle aspiration (FNA) and ultrasound. However, FNA has a limited ability to distinguish between benign and malignant lesions, especially in cases with indeterminate cytology. Although the clinical course of differentiated thyroid carcinoma is believed to be favorable, delayed diagnosis can make its clinical management difficult. Many immunohistochemical (IHC) or molecular adjunctive markers have been tested to improve the diagnostic accuracy for thyroid nodules. The common IHC markers galectin-3, Hector Battifora mesothelial-1, and cytokeratin-19 are used alone or as part of panels for both FNA and analysis of surgical specimens. A novel IHC marker, podoplanin, was recently introduced as an adjunctive marker for thyroid cancer diagnosis and prognosis and is associated with the progression of papillary thyroid carcinoma (PTC). Several researchers have identified molecular markers to increase the diagnostic accuracy of thyroid lesions of undetermined significance. Four promising molecular markers have been proposed and thoroughly investigated: B-type Raf kinase (BRAF) and RAS, rearranged in transformation/PTC (RET/PTC), paired box gene 8 (Pax8)/peroxisome proliferator-activated receptor gamma (PPARγ). BRAF mutations can be measured by immunohistochemistry using an antibody specific to the mutated protein. In this review, we focused on the limitations of current diagnostic tools and on determining the application of the above-mentioned markers to thyroid nodule diagnosis.

Key Words: Differentiated thyroid carcinoma, Immunohistochemistry, Molecular markers

for its propensity of hematic and lymphatic spread, its prognosis is excellent with the proper diagnosis and

Introduction

treatment. Basically, DTC diagnosis largely depends on microscopic examination by a pathologist, which is

Thyroid cancers are rapidly increasing endocrine 1)

malignancies. Thyroid cancers account for 31.1% of

very important to initiate the appropriate treatment. Due

women ’s cancers and 6.4% of men ’s cancers. It is

to its good cost-effectiveness, fine-needle aspiration

estimated that 40,568 individuals (33,562 women and

(FNA) is one of the first diagnostic procedure choices

7,006 men) were diagnosed with thyroid cancer in

in the clinical management of thyroid nodules. Ultra-

2011. Of all thyroid cancers, differentiated thyroid car-

sound (US) associated with FNA cytology is quite im-

cinoma (DTC) is the most common type of follicular

portant to distinguish benign thyroid nodules from ma-

epithelial cell derived tumors. 90% of all thyroid cancers.

4)

2,3)

5)

6)

It accounts for about

lignant nodules. The introduction of FNA in the diag-

Though DTC is famous

nostic procedure increased the selection of adequate

Received November 12, 2014 / Revised January 27, 2015 / Accepted February 2, 2015 Correspondence: Kyu Eun Lee, MD, PhD, Department of Surgery, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea Tel: 82-2-2072-2081, Fax: 82-2-766-3975, E-mail: kyu.eun.lee.md@gmail.com Copyright ⓒ 2015, the Korean Thyroid Association. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

50

Immunohistochemical and Molecular Markers Associated with Differentiated Thyroid Carcinoma

patients for surgical treatment and allowed the pre7)

characteristic nuclear features such as irregular and

vention of unnecessary surgery, because the proce-

enlarged nucleus, optical clearing, fine chromatin,

8)

elongation, overlapping, micronucleoli, nuclear groo-

For the last decade, many studies were designed

ves and pseudoinclusion.4,16,17) However, these cytol-

to improve FNA diagnostic accuracy.9) Many im-

ogy criteria are sometimes not enough to distinguish

munohistochemical (IHC) markers have been propo-

thyroid gland malignancies from benign lesions. Thus,

sed, including galectin-3, Hector Battifora meso-

IHC and molecular markers have been employed to

thelial-1 (HBME-1), cytokeratin-19 (CK-19), and

improve the diagnostic accuracy of FNA cytology. A

novel molecular markers (i.e. B-type Raf kinase

recent systemic review, analyzing cumulative data, re-

[BRAF ]), rearranged in transformation/papillary thyroid

ported that the combination of IHC positivity for ga-

carcinoma (RET/PTC), RAS , and paired box gene 8

lectin-3, HBME-1 and CK-19 as well as galectin-3

(Pax8 )/peroxisome

dure allows about 65-80% of accurate diagnoses.

proliferator-activated

receptor

preoperative expression proved to be very sensitive

2,10,11)

Data sug-

tests to distinguish benign lesions from differentiated

gest that IHC markers improve the performance of

thyroid carcinoma.11) Another review of the literature

FNA cytology in the diagnosis of patients with in-

also supported the use of ancillary techniques, involv-

determinate cytology.1) Additionally, podoplanin, which

ing a panel of antibodies for IHC as well as molecular

is a proven prognosis factor in other cancers such as

analysis in the assessment of thyroid nodules com-

colorectal and breast cancer, has been studied in the

bined with standard morphologic criteria.

gamma (PPAR γ) to diagnose DTC.

3)

prediction of lymph node metastasis in patients with DTC.

12-15)

Galectin-3

This review aimed at not only explaining the general characteristics of these numerous IHC and molecular

Galectin-3 is a subtype of β-galactoside-binding

markers in DTC diagnosis and prognosis but also dis-

lectins identified in the cytoplasm and nucleus.18) It

cussing the individual efficacy of these markers for

plays an important role in cell-cell and cell-matrix in-

diagnosis.

teraction, cell growth, malignant transformation, and apoptosis.9) It is expressed by human macrophages

Immunohistochemical Markers

and neutrophils, mast cells, and Langerhans cells. It is also known to be associated with inflammation, cell

Most thyroid cancers are easily diagnosed by using

damage repair, and metastasis.

conventional histopathologic criteria,3) which include

19,20)

Many studies reported galectin-3 overexpression in

Table 1. Immunohistochemical detection of galectin-3 in thyroid surgical specimens* Normal

CLT

NH

FA

PTC

FTC

PDTC

ATC

23)

Fernandez et al. 0 (0) 0 (0) 0 (0) 18/18 (100) 4/8 (50) 2/3 (67) 5/5 (100) 0 (0) 3/8 (37) 22/34 (64) 2/3 (67) Herrmann et al.19) 20) 7/7 (100) 4/19 (21) 19/20 (95) 7/10 (70) Kovacs et al. 71) 4/13 (31) 22/24 (92) 4/9 (44) Weber et al. 0 (0) 16/29 (55) 2/21 (9) 63/67 (94) 4/6 (67) 4/4 (100) Prasad et al.33) 72) 7/15 (47) 15/18 (83) 7/11 (64) Oestreicher-Kedem et al. 21) 169/202 (84) Cvejic et al. 0/75 (0) 2/4 (50) 0/50 (0) 5/132 (4) 195/201 (97) 54/57 (95) 13/20 (65) 18/20 (90) Bartolazzi et al.22) 29) 3/50 (6) 39/39 (100) 16/19 (84) Saggiorato et al. 9) 3/54 (6) 1/35 (29) 179/181 (99) 16/25 (64) Park et al. *Values are number/total number (percent). Hyphen indicate not determined in this study ATC: anaplastic thyroid carcinoma, CLT: chronic lymphocytic thyroiditis, FA: follicular adenoma, FTC: follicular thyroid carcinoma, NH: nodular hyperplasia, PDTC: poorly differentiated thyroid carcinoma, PTC: papillary thyroid carcinoma

51 Clin Exp Thyroidol

Jun Woo Jung, et al

malignant tumors.3) In a recent study, galectin-3 was

tivity, specificity, positive predictive value and diag-

depicted as the most accurate marker for DTC diag-

nostic accuracy were 100%, 98%, 92%, and 99%,

18)

respectively.22) In contrast, a relatively lower specificity

The reported frequencies of galectin-3 expression in

was estimated in smaller series due to the consid-

DTC are presented in Table 1.

erable expression of galectin-3 in follicular adenoma

nosis when compared with other molecular markers.

Galectin-3 staining shows a strong diffuse cytoplasmic staining in most cases of PTC, including the 3)

(11 out of 44 cases, 25%).

24)

These studies imply that galectin-3 is a useful tool

conventional and follicular variant types (Fig. 1A). Ga-

for DTC diagnosis. However, it should not be used as

lectin-3 is observed in 64-100% of patients with con-

a single diagnostic marker. Clinicians have to be cau-

ventional PTC

21)

and 44-100% of patients with folli3)

tious when interpreting positive results in the absence

cular thyroid carcinoma (FTC) (Fig. 1B). Galectin-3 is

of DTC definite morphologic features, due to the prob-

less frequently detected in follicular adenomas (0-

ability of false positive.3) Similarly, caution should be

37.5%; mean, 21.84%, Fig. 1C).22) Hyperplastic nod-

taken when evaluating unconventional forms of PTC.

ules, nodular goiters, and normal follicular epithelium usually do not express galectin-3.19,22,23) In a large

HBME-1

multicenter study comprising 226 preoperative FNA specimens of thyroid nodules (188 benign lesions and

HBME-1 is a monoclonal antibody reacting against

34 carcinomas), galectin-3 immunodetection sensi-

unknown antigens existing on mesothelial cell sur-

Fig. 1. Galectin-3 immunostaining. (A) Galectin-3 is diffusely expressed in papillary thyroid carcinoma. It is localized in both the cytoplasm and nuclei (×400). (B) Focal expression of galectin-3 is detected in follicular thyroid carcinoma (×200). (C) Galectin-3 is not detected in follicular adenoma (×200). Vol. 8, No. 1, 2015

52


face.25) Originally, HBME-1 was commonly used in

like nodular hyperplasia and follicular adenoma.26-29)

differential diagnosis of mesothelioma and adenocar-

HBME-1 is highly expressed in both PTC and FTC,

17)

Several investigators reported HBME-1 as

but weakly expressed in MTC and anaplastic thyroid

a useful diagnostic marker to differentiate thyroid can-

carcinoma (ATC). It is involved in cancer cell pro-

cers, especially DTC from other benign thyroid lesions

liferation and migration.

cinoma.

25)

Table 2. HBME-1 immunohistochemical detection in thyroid surgical specimens* Normal 33)

Prasad et al. 2) Cheung et al. 31) Mase et al. Choi et al.30) 32) Papotti et al. 29) Saggiorato et al. 73) Nikiforova et al. 9) Park et al.

0/59 (0) -

NH

FA

1/29 (3) 2/21 (10) 0/35 (0) 0/35 (0) 8/62 (12) 17/62 (27) 1/15 (6.6) 2/50 (4) 3/23 (13) 11/54 (20.4) 17/35 (49)

PTC 57/67 (85) 76/138 (55) 35/36 (97.2) 65/67 (97) 14/14 (100) 37/39 (94.8) 166/181 (92)

FTC 3/6 2/4 33/39 30/30

(50) (50) (84.6) (100) 17/19 (89.5) 11/33 (34) 22/25 (88)

PDTC

ATC

4/6 (67) 1/2 (50) 0/2 (0) 11/12 (91.6) 2/10 (20) -

*Values are number/total number (percent). Hyphen indicate not determined in this study ATC: anaplastic thyroid carcinoma, CLT: chronic lymphocytic thyroiditis, FA: follicular adenoma, FTC: follicular thyroid carcinoma, NH: nodular hyperplasia, PDTC: poorly differentiated thyroid carcinoma, PTC: papillary thyroid carcinoma

Fig. 2. HBME-1 immunostaining. (A) Papillary thyroid carcinoma exhibits a strong membranous staining (×200). (B) Follicular thyroid carcinoma exhibits a diffuse cytoplasmic staining (×200). (C) Follicular adenoma shows focal expression of HBME-1 (×200).


Jun Woo Jung, et al

In PTC, most cases show diffuse positive staining for HBME-1 (55-100%; mean, 88%).

2,29,30)

In FTCs,

patterns in either benign or malignant thyroid nodules are shown in Fig. 2.

HBME-1 detection showed a high variability between

A meta-analysis of IHC markers, with a special fo-

studies (50-100%; mean, 75%).2,29-31) A few studies

cus on HBME-1, comprising 3900 samples reported

indicated HBME-1 staining in follicular patterned tu-

that the diagnostic odds ratio (dOR) of HBME-1

mors of uncertain malignant potential that had either

(40.97) was higher than that of galectin-3 (23.41) and

questionable vascular/capsular invasion or incomplete

CK-19 (14.73).11) dOR was estimated using sensitivity

nuclear features of PTCs (29-66% of cases).32,33)

and specificity values directly. This measurement

Many studies reported the absence of HBME-1 in

shows the overall diagnostic power of each individual

2,33)

normal and hyperplastic thyroid.

However, HBME-1

has been detected in cases of adenomatous goiter

marker (a high dOR implies that the test presents a good diagnostic power).

11)

(3-12%) and follicular adenomas (0-27%).2,29,31-33)

HBME-1 positivity has been considered as a useful

HBME-1 sensitivity, specificity, positive predictive val-

adjunctive marker in the discrimination of thyroid le-

ue, and diagnostic accuracy in distinguishing benign

sions using FNA cytology, due to its high sensitivity

from malignant lesions as a single marker are 80%,

and specificity.29,34) However, its prognostic value is still

96%, 96.7%, and 86.4%, respectively.29) The results of

in doubt and further studies are needed.

reported HBME-1 staining in thyroid nodules by IHC are presented in Table 2. Various HBME-1 staining

Fig. 3. CK-19 immunostaining. (A) Papillary thyroid carcinoma exhibits a diffuse expression of CK-19 (×200). (B) Focal expression of CK19 is detected in follicular thyroid carcinoma (×200). (C) CK-19 is not detected in follicular adenoma (× 200). Vol. 8, No. 1, 2015

54


CK-19 CK-19 belongs to the keratin (CK) family (48-60 kDa). It is expressed in simple or glandular epithelium like the thyroid gland.25) Many studies have been designed using various antibodies against CKs to identify the most effective patterns in normal parenchyma, benign nodules, and malignant tumors.3) Among all CKs, CK-19 has become one of the most useful markers to diagnose thyroid nodule lesions. In fact, CK-19 detection in follicular adenomas and other benign nodules is less intense and more focal than in DTC (Fig. 3).

2,35-37)

Many authors stress the importance of the

distribution pattern and intensity of CK-19 staining as

Fig. 4. BRAF staining in papillary thyroid carcinoma. A diffuse expression can be distinctively observed in both the cytoplasm and nuclei (×200).

the most crucial factors for an accurate interpretation.2,36-38) Its intense immunoreactivity might help di-

using IHC.41) They reported that 76 cases (52.8%)

agnose DTC using IHC.39) CK-19 reported sensitivity

showed definite diffuse cytoplasmic expression of the

and specificity when used as a single marker are as

mutated protein and these selected cases were con-

high as 92% and 97%, respectively.40) CK19 can be

firmed by sequencing analysis.41) All patients pre-

a crucial marker along with a panel of other markers

sented the BRAF T1799A point mutation. The authors

due to its high expression in DTC.

25)

However, CK19

concluded that BRAF antibody may be a promising

staining of cytologic specimens should be interpreted

diagnostic marker alone or as part of a panel of mar-

with great caution because of its well-known reactivity

kers. However, BRAF mutation-specific antibody pos-

in atypia and chronic lymphocytic thyroiditis that can

itive staining did not present a high sensitivity in this

be observed in PTCs.

3)

study. Additionally, BRAF mutation-specific antibody positive staining can warrant further gene mutation

BRAF

study. The BRAF antibody staining patterns are presented in Fig. 4.

The V600E mutation of the BRAF gene is a wellknown genetic event in PTC and thought to be in-

Podoplanin

volved in the development and progression of malignancy.41) Various methods have been reported for

Podoplanin, a mucin-type transmembrane glyco-

BRAF mutation analysis with different accuracies, including gene sequencing (Sanger sequencing, pyrosequencing) and polymerase chain reaction (PCR).8,41) However, these methods are somewhat complex and expensive and often complicated by inadequate DNA preservation in formalin-fixed and paraffin-embedded tissues and by the presence of reactive thyroid pa42-44) renchyma. In a recent study, in which 144 patients with PTC were enrolled, the expression of the mutated BRAF V600E protein was evaluated with a novel mutation specific antibody, called clone VE1,

protein specific to the lymphatic channel is expressed


in various human cancers, including colorectal, breast, and pancreas cancer, and is regarded as a factor promoting tumor progression.14) Its expression by cancer-associated fibroblasts is known to be an indicator of poor prognosis in some types of cancer.

15)

Though podoplanin biological functions are not yet completely understood, the available data suggest that it can play an important role in tumor cell invasion.45) Rudzinska et al.,14) using IHC, RT-PCR, and western blotting in cancer cell lines, reported that podoplanin

Jun Woo Jung, et al

is moderately expressed in PTC (40%) and can be

glutamic acid for a valine at position 600 (V600E) has

considered as a pro-metastatic factor involved in PTC

been observed in up to 50% of classic PTCs and 25%

propagation.

of its follicular variant (FVPTC).

51,52)

However, it was reported to be positive in only 16% of thyroid cancers that presented an indeterminate or

Molecular Markers

suspicious FNA cytology and PTC or FVPTC histology.53)

It is well recognized that IHC assessment of thyroid

The prevalence of the BRAF mutation in follicular thy-

nodules presents few limitations, including a large

roid carcinoma is just around 1%.46) Thus, diagnostic

spectrum of sensitivity and specificity, inconsistency in

testing for this mutation cannot serve as an indepen-

the method, and various scoring systems. These limi-

dent cancer diagnostic marker in evaluation of in-

tations prompted us to investigate the diagnostic effi-

determinate nodules.46,53) Nevertheless, it is promising

cacy of molecular markers.46) To date, the three larg-

as part of a panel. Despite a low sensitivity, BRA F mu-

est reported studies are: Nikiforov and colleaguesʼ

tation specificity is quite high with a false positive rate

study

47)

(513 indeterminate nodules), which used

of only 0.2%.

54,55)

BRAF V600E (termed BRAF mutation in this review)

A more recent meta-analysis, summarizing the re-

and RAS mutation genetic tests as well as RET-PTC

sults of 6 studies and including 2800 malignant and

and PAX8/PPAR γ rearrangements; Bartolazzi and

benign lesions, reported a BRAF mutation specificity of

48)

(432 indeterminate nodules),

97.9% and a positive predictive value of 99.9%. Only

which used galectin-3 IHC; and Alexander and col-

seven false positive results were identified in three dif-

leagues ’ study 49) (265 indeterminate nodules), which

ferent studies.

colleagues ’ study

56,57)

In term of prognostic value, a multicenter study sh-

used the 142 gene expression classifier. In this review, we try to focus on the recent reports

owed a strong association of BRAF mutation with

on the above-mentioned molecular markers, BRAF

lymph node metastasis, extrathyroidal extension, man-

and RAS mutations, as well as RET-PTC and PAX8/

ifestation in advanced disease (stages III and IV), and

PPAR γ rearrangements. Table 3 summarized DTC diagnosis efficacy of these four molecular markers in 8) FNA specimens.

recurrence.58) Additionally, BRAF mutation has previously been associated with the loss of radioiodine avidity of recurrent papillary thyroid cancer, making the disease refractory to radioiodine treatment.58) Many studies indicated an association between BRAF muta-

BRAF Mutation

tion and decreased or negative expression of thyroid BRAF is a member of the mitogen-activated protein

iodide-handling genes, including SLC5A5 (also kn-

kinase (MAPK) pathway that is associated with cell

own as NIS ), thyroid stimulating hormone receptor

50)

Thou-

(TSHR ), SLC26A4 (also known as pendrin), TPO, and

gh many different BRAF mutations have been involved

TG.59,60) BRAF mutation has also been shown to lead

in the pathogenesis of several malignancies, a specific

to the overexpression of many tumor-promoting mol-

activating mutation resulting from the substitution of a

ecules such as vascular endothelial growth factor

proliferation, cell differentiation, and apoptosis.

Table 3. Molecular markers used in the preoperative evaluation of thyroid nodule FNA. From Rodrigues et al.

BRAF mutations RAS Mutations RET rearrangements PAX8/PPARγ rearrangements

8)

Average SN

Average SP

Average PPV

Average NPV

Average AC

52.35 23.00 18.20 20.00

97.92 97.20 88.72 100.00

99.85 82.20 87.00 100.00

51.62 63.20 59.60 46.00

70.54 65.00 55.30 60.00

AC: accuracy, NPV: negative predictive value, PPV: positive predictive value, SN: sensitivity, SP: specificity Vol. 8, No. 1, 2015

56


(VEGF) and MET.59,60) These results provide the mo-

DTCs, with a predominant distribution among FTCs

lecular basis for the aggressiveness and treatment

and FVPTCs.51) RAS mutation is considered to be im-

failure of papillary thyroid cancer in association with BRAF mutation 61) and also suggest the strong association of BRAF mutation with papillary thyroid cancer 62) mortality in an international multicenter study.

portant for the detection of FVPTC, which are difficult to diagnose by FNA cytology.1) While there might be some false positive nodules of benign follicular adenomas, it seems that RAS-positive follicular adenomas are precursor lesions for RAS-positive FTCs or FVPTCs and RAS mutation obviously predisposes the

RET/PTC Rearrangement

well-differentiated cancer to dedifferentiation, leading The most common types of chromosomal re-

to a more aggressive cancer.

68)

arrangement in thyroid cancer are the intrachromo-

RAS mutation is not sensitive and specific enough

somal rearrangement like RET/PTC1 and RET/PTC3.55)

to serve as a single marker for prediction of benign

RET/PTC rearrangements are also known to activate

or malignant nodules. Therefore, RAS mutation results

both the MAPK and phosphatidyl-3-inositol/protein

must be interpreted with caution. However, RAS mu-

kinase B (PI3K/Akt) pathways and are responsible for

tational study could prove to be a very powerful ad-

thyroid dedifferentiation.63) RET/PTC rearrangements

junctive tool to cytology, as many cytologically in-

can be useful thyroid cancer diagnostic markers. The

determinate aspirates harbor RAS mutations, suggest-

preoperative diagnosis of thyroid nodules might be

ing the presence of cancer.

46)

more accurate through the detection of RET/PTC in thyroid FNA specimens, in particular in those that pre-

PAX8/PPARγ Rearrangement

sent indeterminate cytology or donʼt have a sufficient amount of cells for cytologic assessment.

64,65)

Rearrangements between PAX8 and PPAR γ ge-

H ow ever, R ET/PTC rearrangem ents have also

nes, PAX8 / PPAR γ rearrangements, a kind of fusion

been detected in follicular adenom a and other

oncogene, are mostly found in follicular tumors (30-

benign lesions at m odest rates that lim it their utility

40% of FTCs and 2-10% of follicular adenomas) and

66)

A recent m eta-

are rare in non-classical PTC, especially in FVPTC

analysis indicates that R ET/P TC specificity aver-

(＜5%).69) Though the mechanism by which this gene

age value is 18% and its positive predictive value

rearrangement influences malignant transformation is

as single diagnostic m arkers.

is 87% .

11)

R ET/P TC rearrangem ents are relatively

not fully understood, it is assumed to be associated

rare com pared to BRAF or RAS m utation. It is im-

with inhibition of the antiproliferative property of the

portant not to overestim ate the value of R ET/P TC

PPARγ receptor.46) Many investigators suggested that

gene rearrangem ents as a single m arker and

such oncogenic activation subsequently causes the

caution

affected nodule to show a malignant behavior. Thus,

should

be

taken

for

thyroid

cancer

such lesions should be treated as precancerous.46)

diagnosis.

However, Sahin et al.70) reported that tumors with

RAS Mutation

PAX8 / PPAR γ rearrangements usually carry a favorable prognosis.

The RAS oncogene family consists of 3 genes (HRAS, KRAS , and NRAS) that encode small GTPase proteins members of signal transduction.46) Activating

Conclusion

mutations in these genes stimulate the MAPK and

The accurate diagnosis of thyroid nodule is the cor-

PI3K/Akt pathways regulating cell growth, proliferation,

nerstone for the determination of the appropriate treat-

differentiation, mobility, and mortality.

67)

This mutation may be detected in up to 40% of 57 Clin Exp Thyroidol

ment for patients. It is very important in many clinical aspects, such as operability, complication rate related

Jun Woo Jung, et al

to surgery, prognosis, and patient quality of life. Even though histopathology and FNA cytology are commonly used to diagnose thyroid nodule, they present limitations, which have been summarized in previous studies. Thus, IHC and molecular markers can play a key role in the proper management of thyroid cancer. These modalities combined with conventional diagnostic tools will improve thyroid cancer diagnostic accuracy. Furthermore, testing FNA samples using panels of markers, including several IHC and molecular markers, may allow us to increase the diagnostic accuracy. Today, the cost of these tests continues to drop, which will make the use of IHC and molecular markers easier in a close future. Though they have some limitations such as uneven cost in many different countries, inconsistent sensitivity and specificity, large variation in performance, cost-effectiveness of IHC and molecular markers has been proven in recent study.61) They have shown a favorable profile, despite these limitations, and IHC is usually cheaper than molecular and other genomic tests. It might be more useful in low-income countries. Besides the diagnostic utility of molecular markers, the elucidation of thyroid cancer molecular basis will improve the therapeutic outcomes.

Conflict of Interest The authors have no conflict of interest to declare.

Acknowledgments This work was supported by grant no. 04-20070320 and no. 04-2011-1090 from the SNUH Research Fund.

References 1) Fallahi P, Giannini R, Miccoli P, Antonelli A, Basolo F. Molecular diagnostics of fine needle aspiration for the presurgical screening of thyroid nodules. Curr Genomics 2014; 15(3):171-7. 2) Cheung CC, Ezzat S, Freeman JL, Rosen IB, Asa SL. Immunohistochemical diagnosis of papillary thyroid carcinoma. Mod Pathol 2001;14(4):338-42. 3) Fischer S, Asa SL. Application of immunohistochemistry to

thyroid neoplasms. Arch Pathol Lab Med 2008;132(3):359-72. 4) Griffith OL, Chiu CG, Gown AM, Jones SJ, Wiseman SM. Biomarker panel diagnosis of thyroid cancer: a critical review. Expert Rev Anticancer Ther 2008;8(9):1399-413. 5) Singer PA, Cooper DS, Daniels GH, Ladenson PW, Greenspan FS, Levy EG, et al. Treatment guidelines for patients with thyroid nodules and well-differentiated thyroid cancer. American Thyroid Association. Arch Intern Med 1996;156(19):2165-72. 6) American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009;19(11):1167-214. 7) Gasbarri A, Marchetti C, Iervasi G, Bottoni A, Nicolini A, Bartolazzi A, et al. From the bench to the bedside. Galectin-3 immunodetection for improving the preoperative diagnosis of the follicular thyroid nodules. Biomed Pharmacother 2004;58(6-7): 356-9. 8) Rodrigues HG, de Pontes AA, Adan LF. Use of molecular markers in samples obtained from preoperative aspiration of thyroid. Endocr J 2012;59(5):417-24. 9) Park YJ, Kwak SH, Kim DC, Kim H, Choe G, Park DJ, et al. Diagnostic value of galectin-3, HBME-1, cytokeratin 19, high molecular weight cytokeratin, cyclin D1 and p27(kip1) in the differential diagnosis of thyroid nodules. J Korean Med Sci 2007;22(4):621-8. 10) Vriens MR, Schreinemakers JM, Suh I, Guerrero MA, Clark OH. Diagnostic markers and prognostic factors in thyroid cancer. Future Oncol 2009;5(8):1283-93. 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 2012;7:97. 12) Yasuoka H, Nakamura Y, Zuo H, Tang W, Takamura Y, Miyauchi A, et al. VEGF-D expression and lymph vessels play an important role for lymph node metastasis in papillary thyroid carcinoma. Mod Pathol 2005;18(8):1127-33. 13) Scarpino S, Di Napoli A, Melotti F, Talerico C, Cancrini A, Ruco L. Papillary carcinoma of the thyroid: low expression of NCAM (CD56) is associated with downregulation of VEGF-D production by tumour cells. J Pathol 2007;212(4):411-9. 14) Rudzinska M, Gawel D, Sikorska J, Karpinska KM, Kiedrowski M, Stepien T, et al. The role of podoplanin in the biology of differentiated thyroid cancers. PLoS One 2014;9(5): e96541. 15) Shindo K, Aishima S, Ohuchida K, Fujino M, Mizuuchi Y, Hattori M, et al. Podoplanin expression in the cyst wall correlates with the progression of intraductal papillary mucinous neoplasm. Virchows Arch 2014;465(3):265-73. 16) Hapke MR, Dehner LP. The optically clear nucleus. A reliable sign of papillary carcinoma of the thyroid? Am J Surg Pathol 1979;3(1):31-8. 17) Miettinen M, Karkkainen P. Differential reactivity of HBME1 and CD15 antibodies in benign and malignant thyroid tumours. Preferential reactivity with malignant tumours. Virchows Vol. 8, No. 1, 2015

58

Immunohistochemical and Molecular Markers Associated with Differentiated Thyroid Carcinoma Arch 1996;429(4-5):213-9. 18) Chiu CG, Strugnell SS, Griffith OL, Jones SJ, Gown AM, Walker B, et al. Diagnostic utility of galectin-3 in thyroid cancer. Am J Pathol 2010;176(5):2067-81. 19) Herrmann ME, LiVolsi VA, Pasha TL, Roberts SA, Wojcik EM, Baloch ZW. Immunohistochemical expression of galectin-3 in benign and malignant thyroid lesions. Arch Pathol Lab Med 2002;126(6):710-3. 20) Kovacs RB, Foldes J, Winkler G, Bodo M, Sapi Z. The investigation of galectin-3 in diseases of the thyroid gland. Eur J Endocrinol 2003;149(5):449-53. 21) Cvejic DS, Savin SB, Petrovic IM, Paunovic IR, Tatic SB, Havelka MJ. Galectin-3 expression in papillary thyroid carcinoma: relation to histomorphologic growth pattern, lymph node metastasis, extrathyroid invasion, and tumor size. Head Neck 2005;27(12):1049-55. 22) Bartolazzi A, Gasbarri A, Papotti M, Bussolati G, Lucante T, Khan A, et al. Application of an immunodiagnostic method for improving preoperative diagnosis of nodular thyroid lesions. Lancet 2001;357(9269):1644-50. 23) Fernandez PL, Merino MJ, Gomez M, Campo E, Medina T, Castronovo V, et al. Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue. J Pathol 1997; 181(1):80-6. 24) Maruta J, Hashimoto H, Yamashita H, Yamashita H, Noguchi S. Immunostaining of galectin-3 and CD44v6 using fine-needle aspiration for distinguishing follicular carcinoma from adenoma. Diagn Cytopathol 2004;31(6):392-6. 25) Sethi K, Sarkar S, Das S, Mohanty B, Mandal M. Biomarkers for the diagnosis of thyroid cancer. J Exp Ther Oncol 2010; 8(4):341-52. 26) Wallander M, Layfield LJ, Jarboe E, Emerson L, Liu T, Thaker H, et al. Follicular variant of papillary carcinoma: reproducibility of histologic diagnosis and utility of HBME-1 immunohistochemistry and BRAF mutational analysis as diagnostic adjuncts. Appl Immunohistochem Mol Morphol 2010; 18(3):231-5. 27) Erickson LA, Lloyd RV. Practical markers used in the diagnosis of endocrine tumors. Adv Anat Pathol 2004;11(4):175-89. 28) Rezk S, Khan A. Role of immunohistochemistry in the diagnosis and progression of follicular epithelium-derived thyroid carcinoma. Appl Immunohistochem Mol Morphol 2005;13(3): 256-64. 29) Saggiorato E, De Pompa R, Volante M, Cappia S, Arecco F, Dei Tos AP, et al. Characterization of thyroid 'follicular neoplasms' in fine-needle aspiration cytological specimens using a panel of immunohistochemical markers: a proposal for clinical application. Endocr Relat Cancer 2005;12(2):305-17. 30) Choi YL, Kim MK, Suh JW, Han J, Kim JH, Yang JH, et al. Immunoexpression of HBME-1, high molecular weight cytokeratin, cytokeratin 19, thyroid transcription factor-1, and E-cadherin in thyroid carcinomas. J Korean Med Sci 2005; 20(5):853-9. 31) Mase T, Funahashi H, Koshikawa T, Imai T, Nara Y, Tanaka Y, et al. HBME-1 immunostaining in thyroid tumors especially in follicular neoplasm. Endocr J 2003;50(2):173-7. 32) Papotti M, Rodriguez J, De Pompa R, Bartolazzi A, Rosai


33)

34)

35)

36)

37)

38) 39)

40)

41)

42)

43)

44)

45) 46)

47)

J. Galectin-3 and HBME-1 expression in well-differentiated thyroid tumors with follicular architecture of uncertain malignant potential. Mod Pathol 2005;18(4):541-6. 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 thyroid tumors. Mod Pathol 2005; 18(1):48-57. Rossi ED, Raffaelli M, Minimo C, Mule A, Lombardi CP, Vecchio FM, et al. Immunocytochemical evaluation of thyroid neoplasms on thin-layer smears from fine-needle aspiration biopsies. Cancer 2005;105(2):87-95. Cerilli LA, Mills SE, Rumpel CA, Dudley TH, Moskaluk CA. Interpretation of RET immunostaining in follicular lesions of the thyroid. Am J Clin Pathol 2002;118(2):186-93. Baloch ZW, Abraham S, Roberts S, LiVolsi VA. Differential expression of cytokeratins in follicular variant of papillary carcinoma: an immunohistochemical study and its diagnostic utility. Hum Pathol 1999;30(10):1166-71. Sahoo S, Hoda SA, Rosai J, DeLellis RA. Cytokeratin 19 immunoreactivity in the diagnosis of papillary thyroid carcinoma: a note of caution. Am J Clin Pathol 2001;116(5):696702. Asa SL. My approach to oncocytic tumours of the thyroid. J Clin Pathol 2004;57(3):225-32. Rorive S, Eddafali B, Fernandez S, Decaestecker C, Andre S, Kaltner H, et al. Changes in galectin-7 and cytokeratin-19 expression during the progression of malignancy in thyroid tumors: diagnostic and biological implications. Mod Pathol 2002; 15(12):1294-301. Nasser SM, Pitman MB, Pilch BZ, Faquin WC. Fine-needle aspiration biopsy of papillary thyroid carcinoma: diagnostic utility of cytokeratin 19 immunostaining. Cancer 2000;90(5): 307-11. Koperek O, Kornauth C, Capper D, Berghoff AS, Asari R, Niederle B, et al. Immunohistochemical detection of the BRAF V600E-mutated protein in papillary thyroid carcinoma. Am J Surg Pathol 2012;36(6):844-50. Kim HS, Kim JO, Lee DH, Lee HC, Kim HJ, Kim JH, et al. Factors influencing the detection of the BRAF T1799A mutation in papillary thyroid carcinoma. Oncol Rep 2011; 25(6):1639-44. Kim SK, Kim DL, Han HS, Kim WS, Kim SJ, Moon WJ, et al. Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules. Diagn Mol Pathol 2008;17(2):118-25. Lima J, Trovisco V, Soares P, Maximo V, Magalhaes J, Salvatore G, et al. BRAF mutations are not a major event in post-Chernobyl childhood thyroid carcinomas. J Clin Endocrinol Metab 2004;89(9):4267-71. Wicki A, Christofori G. The potential role of podoplanin in tumour invasion. Br J Cancer 2007;96(1):1-5. Kim MI, Alexander EK. Diagnostic use of molecular markers in the evaluation of thyroid nodules. Endocr Pract 2012; 18(5):796-802. Nikiforov YE, Ohori NP, Hodak SP, Carty SE, LeBeau SO, Ferris RL, et al. Impact of mutational testing on the diagnosis

Jun Woo Jung, et al

48)

49)

50) 51)

52)

53)

54) 55)

56)

57)

58)

59)

60) 61)

and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab 2011;96(11):3390-7. Bartolazzi A, Orlandi F, Saggiorato E, Volante M, Arecco F, Rossetto R, et al. Galectin-3-expression analysis in the surgical selection of follicular thyroid nodules with indeterminate fineneedle aspiration cytology: a prospective multicentre study. Lancet Oncol 2008;9(6):543-9. Alexander EK, Kennedy GC, Baloch ZW, Cibas ES, Chudova D, Diggans J, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med 2012; 367(8):705-15. Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 2004;6(4):313-9. Xing M. Genetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancer. Thyroid 2010;20(7): 697-706. Kebebew E, Weng J, Bauer J, Ranvier G, Clark OH, Duh QY, et al. The prevalence and prognostic value of BRAF mutation in thyroid cancer. Ann Surg 2007;246(3):466-70; discussion 470-1. Cohen Y, Rosenbaum E, Clark DP, Zeiger MA, Umbricht CB, Tufano RP, et al. Mutational analysis of BRAF in fine needle aspiration biopsies of the thyroid: a potential application for the preoperative assessment of thyroid nodules. Clin Cancer Res 2004;10(8):2761-5. Nikiforova MN, Nikiforov YE. Molecular diagnostics and predictors in thyroid cancer. Thyroid 2009;19(12):1351-61. Kouniavsky G, Zeiger MA. The quest for diagnostic molecular markers for thyroid nodules with indeterminate or suspicious cytology. J Surg Oncol 2012;105(5):438-43. Chung KW, Yang SK, Lee GK, Kim EY, Kwon S, Lee SH, et al. Detection of BRAFV600E mutation on fine needle aspiration specimens of thyroid nodule refines cyto-pathology diagnosis, especially in BRAF600E mutation-prevalent area. Clin Endocrinol (Oxf) 2006;65(5):660-6. Kim SK, Hwang TS, Yoo YB, Han HS, Kim DL, Song KH, et al. Surgical results of thyroid nodules according to a management guideline based on the BRAF(V600E) mutation status. J Clin Endocrinol Metab 2011;96(3):658-64. Xing M, Westra WH, Tufano RP, Cohen Y, Rosenbaum E, Rhoden KJ, et al. BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005;90(12):6373-9. Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 2007;28(7):742-62. Xing M. Prognostic utility of BRAF mutation in papillary thyroid cancer. Mol Cell Endocrinol 2010;321(1):86-93. Xing M, Haugen BR, Schlumberger M. Progress in molecular-based management of differentiated thyroid cancer. Lancet

2013;381(9871):1058-69. 62) Xing M, Alzahrani AS, Carson KA, Viola D, Elisei R, Bendlova B, et al. Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 2013;309(14):1493-501. 63) Ciampi R, Nikiforov YE. RET/PTC rearrangements and BRAF mutations in thyroid tumorigenesis. Endocrinology 2007;148(3): 936-41. 64) Cheung CC, Carydis B, Ezzat S, Bedard YC, Asa SL. Analysis of ret/PTC gene rearrangements refines the fine needle aspiration diagnosis of thyroid cancer. J Clin Endocrinol Metab 2001; 86(5):2187-90. 65) Pizzolanti G, Russo L, Richiusa P, Bronte V, Nuara RB, Rodolico V, et al. Fine-needle aspiration molecular analysis for the diagnosis of papillary thyroid carcinoma through BRAF V600E mutation and RET/PTC rearrangement. Thyroid 2007; 17(11):1109-15. 66) Ishizaka Y, Kobayashi S, Ushijima T, Hirohashi S, Sugimura T, Nagao M. Detection of retTPC/PTC transcripts in thyroid adenomas and adenomatous goiter by an RT-PCR method. Oncogene 1991;6(9):1667-72. 67) Repasky GA, Chenette EJ, Der CJ. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol 2004;14(11):639-47. 68) Basolo F, Pisaturo F, Pollina LE, Fontanini G, Elisei R, Molinaro E, et al. N-ras mutation in poorly differentiated thyroid carcinomas: correlation with bone metastases and inverse correlation to thyroglobulin expression. Thyroid 2000;10(1): 19-23. 69) Marques AR, Espadinha C, Catarino AL, Moniz S, Pereira T, Sobrinho LG, et al. Expression of PAX8-PPAR gamma 1 rearrangements in both follicular thyroid carcinomas and adenomas. J Clin Endocrinol Metab 2002;87(8):3947-52. 70) Sahin M, Allard BL, Yates M, Powell JG, Wang XL, Hay ID, et al. PPARgamma staining as a surrogate for PAX8/PPAR gamma fusion oncogene expression in follicular neoplasms: clinicopathological correlation and histopathological diagnostic value. J Clin Endocrinol Metab 2005;90(1):463-8. 71) Weber KB, Shroyer KR, Heinz DE, Nawaz S, Said MS, Haugen BR. The use of a combination of galectin-3 and thyroid peroxidase for the diagnosis and prognosis of thyroid cancer. Am J Clin Pathol 2004;122(4):524-31. 72) Oestreicher-Kedem Y, Halpern M, Roizman P, Hardy B, Sulkes J, Feinmesser R, et al. Diagnostic value of galectin-3 as a marker for malignancy in follicular patterned thyroid lesions. Head Neck 2004;26(11):960-6. 73) Nikiforova MN, Lynch RA, Biddinger PW, Alexander EK, Dorn GW 2nd, Tallini G, et al. RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma. J Clin Endocrinol Metab 2003;88(5):2318-26.

Vol. 8, No. 1, 2015

60