Characterization of thyroid 'follicular neoplasms' in fine-needle aspiration cytological specimens...

Characterization of thyroid ‘follicularneoplasms’ in fine-needle aspirationcytological specimens using a panel ofimmunohistochemical markers:a proposal for clinical application

E Saggiorato, R De Pompa1, M Volante2, S Cappia1, F Arecco, A P Dei Tos3,F Orlandi and M Papotti1

Section of Endocrinology, Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10,

10043 Orbassano (TO), Italy1Division of Pathology, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy2Department of Biomedical Sciences and Oncology, University of Turin, Turin, Italy3Department of Pathology, Regional Hospital, Treviso, Italy

(Requests for offprints should be addressed to E Saggiorato; Email: [email protected])

Abstract

The distinction of benign from malignant follicular thyroid neoplasms remains a difficult task indiagnostic fine-needle aspiration cytology, and some discrepant results have been reported for theindividual immunocytochemical markers of malignancy proposed so far. The aim of this study wasto test if the combined use of a panel of markers could improve the diagnostic accuracy in thepreoperative cytological evaluation of ‘follicular neoplasms’ in an attempt to reduce the number ofthyroidectomies performed for benign lesions. The immunocytochemical expression of galectin-3,HBME-1, thyroperoxidase, cytokeratin-19 and keratan-sulfate was retrospectively analyzed in 125consecutive fine-needle aspiration samples (cell blocks) of indeterminate diagnoses of ‘follicularthyroid neoplasm’, and compared with their corresponding surgical specimens, including 33 folli-cular carcinomas, 42 papillary carcinomas and 50 follicular adenomas. Statistical analysis on eachmarker confirmed that galectin-3 and HBME-1 were the most sensitive (92% and 80% respectively)and specific (94% and 96% respectively) molecules. The use of these two markers sequentially innon-oncocytic lesions (testing HBME-1 as a second marker whenever galectin-3 proved negative)increased the sensitivity and specificity up to 97% and 95% respectively. In oncocytic lesions,HBME-1 proved to be less sensitive, and the sequential combination of galectin-3 and cytokeratin-19reached 100% of both specificity and sensitivity. Our data showed that, as compared with the useof single markers, the sequential combination of two markers represents the most accurateimmunohistochemical panel in managing patients with a fine-needle aspiration biopsy diagnosis of‘follicular neoplasms’, especially in otherwise controversial categories such as oncocytic tumours.The combination of three or more markers did not substantially improve the diagnostic accuracy ofthe test.

Endocrine-Related Cancer (2005) 12 305–317

Introduction

Fine-needle aspiration biopsy (FNAB) is a well-

established diagnostic technique for the preoperative

evaluation of thyroid nodules, allowing a significant

reduction in the number of surgical operations

(Mazzaferri 1993, Hegedus et al. 2003). However, an

important limitation of FNAB is the lack of sensitivity

in the evaluation of ‘follicular neoplasms’, due to its

inability to differentiate benign (follicular adenomas)


Endocrine-Related Cancer (2005) 12 305–317 DOI:10.1677/erc.1.009441351-0088/05/012–305g 2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

from malignant (follicular carcinomas) lesions (Baloch

et al. 2002, Castro & Gharib 2003). Moreover, the

cytological diagnosis of a follicular variant of papillary

carcinoma represents an additional problem, since the

diagnostic cytological criteria of papillary carcinoma

(cytoplasmic invaginations into the nucleus or abun-

dant nuclear grooves) may be lacking (in such cases

the nuclei show coarse chromatin similar to those of

follicular carcinoma) or not completely unequivocal

(features suggestive but not diagnostic of papillary

carcinomas), as also reported in histological practice

(Lloyd et al. 2004); thus the tumour is mainly inclined

to be cytologically interpreted as a ‘follicular neoplasm’

(Kini 1987).

To date, no effective methods have been established

to identify preoperatively malignant follicular tumours

of the thyroid.

Although several additional procedures, such as

advanced image systems (Baloch et al. 2000, Harms

et al. 2002, Papini et al. 2002) and modified techniques

for thyroid biopsy (Tangpricha et al. 2001), have been

developed to increase the FNAB diagnostic accuracy

of thyroid lesions, nowadays none of them has led

to any significant improvement in solving the ‘folli-

cular neoplasm’ dilemma (Yang et al. 2003). Thus, the

identification of new diagnostic approaches is of para-

mount importance in order to provide new criteria for

an early preoperative diagnosis, and reduce the diag-

nostic category of ‘indeterminate’ FNABs.

To this purpose, recent advances in molecular

diagnostics, such as immunocytochemistry, enzyme

activity assays and RT-PCR, allowed a further analysis

of FNAB material in an attempt to differentiate benign

from malignant thyroid nodules (Haugen et al. 2002).

Several molecules involved in carcinogenic processes

have been proposed as markers of thyroid malignancy

(oncogene products, altered enzymes, integrins, cad-

herins, lectins, etc.) (Ringel 2000, Serini et al. 1996,

Nikiforova et al. 2003, Cohen et al. 2004). Among

them, telomerase (Saji et al. 1999, Liou et al. 2003),

high mobility group proteins I(Y) (HMGI(Y)) (Chiap-

petta et al. 1998), the cell surface mesothelial antigen

HBME-1 (Sack et al. 1997, van Hoeven et al. 1998,

Prasad et al. 2004), thyroperoxidase (TPO) (Henry

et al. 1994), cytokeratin-19 (CK19) (Khurana et al.

2003) and galectin-3 (GAL3) (Bartolazzi et al. 2001,

Lloyd 2001) seem to be the most promising molecules

in significantly increasing FNAB accuracy. The mole-

cular detection of specific alterations, mainly restricted

to papillary carcinoma, such as BRAF mutations and

RET/PTC translocations, have also been proposed in

FNAB samples (Cheung et al. 2001a, Cohen et al.

2004, Salvatore et al. 2004) but, although they may

represent promising future methods, they are not easily

accessible to most routine laboratories.

GAL3 polypeptide is a member of the

oligosaccaride-selective binding protein family known

as lectins (Barondes et al. 1994). GAL3 plays important

roles in cell–cell and cell–matrix interactions, extra-

cellular matrix organization, mRNA splicing, cell

growth and apoptosis, neoplastic transformation and

metastasization (Liu et al. 2002, Yang & Liu 2003). As

reported by several studies, this lectin is expressed in

thyroid carcinoma but not in normal thyrocytes and

in benign lesions such as follicular adenoma (Xu

et al. 1995, Fernandez et al. 1997). In particular, we

previously reported that cytoplasmic GAL3, selectively

expressed in malignant thyroid cells, is easily detectable

also on FNAB cytological samples (Orlandi et al. 1998,

Bartolazzi et al. 2001).

The HBME-1 antigen, which is recognized by the

monoclonal antibody HBME-1 (Sheibani et al. 1992),

was shown to be a useful marker of follicular-derived

malignant tumours of the thyroid in both surgical and

FNAB cytological samples (Miettinen & Karkkainen

1996, Sack et al. 1997, van Hoeven et al. 1998, Cheung

et al. 2001b).

TPO is an enzyme present in all non-malignant

thyroid follicular cells. It is immunologically altered

in cancer cells and one of the monoclonal antibodies

raised against intact TPO (MoAB47) fails to recognize

the TPO forms of malignant thyroid tumours (De

Micco et al. 1991). Some studies have shown that

TPO immunostaining highly improves the FNAB

diagnostic accuracy of thyroid follicular lesions (Henry

et al. 1994, Christensen et al. 2000, Raffaelli et al. 2001,

Weber et al. 2004).

CK19 is a low molecular weight cytokeratin widely

present in simple epithelia and in basal cell layers

of stratified epithelium (Rapheal et al. 1994, Porter &

Lane 2003). In both FNAB cytological and surgical

samples of thyroid tumours, CK19 has been found to

be strongly and diffusely expressed in papillary carci-

noma, whereas it is heterogeneously expressed in

follicular carcinomas and absent or focally expressed

in follicular adenomas (Schroder et al. 1996, Fonseca

et al. 1997, Miettinen et al. 1997, Beesley & McLaren

2002, Khurana et al. 2003).

Keratan-sulphate (KS) is a glycoprotein complex

having an unusual relative ratio of highly sulphated

disaccharides and carrying a novel non-characterized

oligosaccharide moiety at the non-reducing terminus,

which is recognized by the monoclonal antibody

373E1 (Magro et al. 2003). This aberrant glycoprotein

complex has been found almost exclusively in thyroid

papillary carcinomas. Moreover, it has been reported

Saggiorato et al.: Application of immunohistochemical markers to thyroid FNAB

306 www.endocrinology-journals.org

that papillary carcinoma-specific KS-bearing macro-

molecules are unique glycoforms of thyroglobulin and

transferrin (Magro et al. 2003). The peculiar KS epi-

tope has been proposed as a novel marker for the

differential diagnosis of benign and malignant thyroid

tumours (Magro et al. 2003).

However, based on the data in the literature, it is

clear that no ‘magic marker’ that distinguishes folli-

cular adenomas from carcinomas has yet been found

(Baloch & LiVolsi 2002). In fact, several reports on

this topic have provided discrepant results. GAL3 was

found in the hands of ourselves and others (Gasbarri

et al. 1999, Inohara et al. 1999, Saggiorato et al. 2001,

2004, Papotti et al. 2002, Volante et al. 2004b, Weber

et al. 2004) to be a highly sensitive marker, but this

was not confirmed by other authors (Nascimento et al.

2001, Martins et al. 2002, Niedziela et al. 2002, Takano

et al. 2003, Mehrotra et al. 2004). The same holds

true for HBME-1 and CK19 (Sahoo et al. 2001, Mai

et al. 2002). Many of these discrepancies stem from

the apparently false-positive staining of some of these

markers in normal thyroid or adenomas (Bartolazzi

et al. 2003, Saggiorato et al. 2004).

In the attempt to solve the aforementioned issues,

we designed this study on a relatively large number

of histologically proven thyroid follicular lesions,

and tested a panel of immunohistochemical markers

(GAL3, HBME-1, TPO, CK19 and KS) on both

FNAB cell blocks and all the corresponding surgical

specimens. The goal was not only to validate the role

of each individual molecule, but also to establish if

any combination of these selected markers could be

helpful in identifying malignant tumours with higher

accuracy in the indeterminate subset of FNABs, thus

reducing the number of thyroidectomies for benign

follicular lesions. In this respect, we showed that the

combination of GAL3 and HBME-1 for conventional

follicular lesions, or GAL3 and CK19 in the case of

oncocytic lesions, provides the highest sensitivity in

FNAB samples as compared with a much larger (and

expensive) panel of markers. Furthermore, we have

developed a clinico-pathological algorithm for the

management of patients with an indeterminate FNAB,

based on the results of these two marker combination

analyses.

Materials and methods

Cytological and histological samples

From the pathology files of San Luigi Hospital

(Orbassano, Turin, Italy) and San Giovanni Battista

Hospital (Turin, Italy), in the years 2002–2004, 125

consecutive cases of FNAB of the thyroid were

collected, in which a diagnosis of ‘follicular neoplasm’

was made, and the subsequent surgical specimens were

filed in the same hospitals and were available for

review.

At operation, all patients had a histological diagnosis

of follicular adenoma or well-differentiated thyroid

carcinoma. The preoperative FNAB diagnosis was:

(i) ‘follicular neoplasm’ (not otherwise specified) in

73/125 samples, histologically classified as follicular

adenomas (43 cases), follicular carcinomas (19 cases)

and follicular variant of papillary carcinomas (11 cases);

(ii) ‘oncocytic cell neoplasm’ in 24/125 specimens,

histologically proven as oncocytic adenomas (seven

cases), oncocytic carcinomas (14 cases) and oncocytic

(follicular) variant of papillary carcinomas (three cases);

(iii) ‘follicular/papillary neoplasm’ (suspected papillary

carcinoma) in 28/125 samples, histologically confirmed

as pure follicular variants (14 cases), or having minor

classical (12 cases) or solid/trabecular (two cases)

components. The whole series included 22 males and

103 females, with a median age of 46 years (range

11–89 years). Written informed consent was obtained

from each patient, and the study was approved by the

San Luigi Hospital review board.

FNABs

Preoperative FNABs were performed with a 22 gauge

needle attached to a 30ml plastic syringe. The

aspirated fluid was in part expelled and smeared onto

charged slides, fixed and stained with a rapid haemato-

xylin and eosin (H&E) method for the evaluation of

adequacy. The remaining material was used to prepare

alcohol-fixed cell blocks (43). Sections of cell block

serial to those employed for H&E routine evaluation

were used for immunohistochemistry.

Surgical specimens

The corresponding surgical samples of all patients

were formalin fixed and paraffin embedded for routine

histopathological diagnosis and immunohistochemical

staining.

Histopathological classification

For all the 125 FNAB cases included in the study,

histopathological review and classification of the corre-

sponding surgical samples was performed, according to

WHO thyroid tumour classification (De Lellis et al.

2004). From three to ten H&E-stained slides as well

as a paraffin block representative of the lesion were

available for review and for the immunocytochemical


www.endocrinology-journals.org 307

procedure. A total of 50 follicular adenomas, 33

follicular carcinomas and 42 papillary carcinomas with

a predominant degree of follicular pattern of growth

were examined. Follicular adenomas consisted of 32

microfollicular, 11 trabecular/solid and seven onco-

cytic cell types whereas, in the follicular carcinoma

group, 14 had oncocytic features and 19 were conven-

tional follicular carcinomas (with a minor trabecular

or insular component in six of them). Among papillary

carcinomas, we observed 25 follicular variants, three

oncocytic follicular variants and 14 cases of follicular

variant with minor classical (12 cases) and solid/

trabecular (two cases) features. Serial sections of a

representative block were obtained from all cases for

immunohistochemistry.

Antibodies

The primary monoclonal antibodies used in this study

(with their working dilutions) were as follows: purified

9C4 to GAL3 (1 : 400; Novocastra Laboratories Ltd,

Newcastle, Tyne and Wear, UK), HBME-1 to HBME-

1 antigen and MoAb47 to TPO (1 : 500 and 1 : 200

respectively; DakoCytomation, Glostrup, Denmark),

b170 to CK19 (1 : 150; Novocastra Laboratories Ltd)

and 373E1 to KS (1 : 100; LabVision Corporation,

Fremont, CA, USA).

Immunoperoxidase technique

Both FNAB cytological cell blocks and surgical

specimens were cut at 4mm thickness, overlaid on

poly-L-lysine-coated slides, dewaxed in xylene, rehy-

drated in decreasing ethanol concentrations and incu-

bated for 10min in phosphate-buffered saline (PBS)

(pH7.4). A heat-induced antigen retrieval procedure

was carried out for all the antigens by placing slides

in 0.01M sodium citrate buffer (pH6.0) (for GAL3,

HBME-1 and KS) and in 0.01M EDTA buffer (pH8.0)

(for TPO and CK19) in a microwave oven set at high

power for three consecutive cycles of 5min each. These

slides were left to cool for 20min at room temperature

and rinsed in PBS. Endogenous peroxidase activity

was quenched with methanol-hydrogen peroxide (3%)

for 15min at room temperature. Slides were then

washed twice in PBS for 5min. Cell blocks and tissue

sections were then incubated in the blocking solution

(ChemMate Buffer Kit; DakoCytomation), and sub-

sequently with the primary antibodies (see above) in a

humidified chamber at room temperature for 45min

(HBME-1 and CK19), or at 4 xC overnight (GAL3,

TPO and KS). After a prolonged wash in PBS, the

sections were incubated with an enzyme-labelled

polymer pre-conjugated with anti-mouse secondary

antibodies (a biotin-free detection system) (Envision

System; DakoCytomation) at room temperature for

30min. The sections were finally washed three times

in PBS, and incubated with 3k-3k-diaminobenzidine-

tetrahydrochloride for 10min. Slides were subsequently

rinsed in tap water, counterstained with Mayer’s

haemalum solution, mounted in Entellan (Merck,

Darmstadt, Germany) and examined with a DM-

RBE Leica photomicroscope (Leica Microsystems AG,

Wetzlar, Germany). Positive controls were histiocytes

for GAL3, a case of mesothelioma for HBME-1,

normal thyroid follicles for TPO, skin for CK19 and

a lymph node metastasis of a papillary thyroid carci-

noma for KS. Negative controls were obtained by

omitting the primary antibody.

Scoring of staining and statistical evaluation

GAL3, HBME-1, TPO, CK19 and KS immunostain-

ings were evaluated blindly by three independent

observers (E S, MP and RDP) without knowledge

of the previously established histological diagnosis,

using the following semiquantitative scale: -no re-

activity, +/- focal reactivity,<10% of the neoplastic

cells,+ moderate reactivity, 10–50% of the neoplastic

cells and ++ diffuse positivity, >50% of the neo-

plastic cells.

Immunostainings for GAL3 (cytoplasmic expression

only), HBME-1, CK19 and KS were considered posi-

tive in the presence of o10% immunoreactive neo-

plastic cells, whereas for TPO a positive case had

>50% neoplastic cells stained.

Sensitivity, specificity, positive/negative predictive

values and diagnostic accuracy of each marker were

calculated as previously described (Orlandi et al. 1998),

and their sequential and simultaneous combinations

were compared. The overall sensitivity and specifi-

city of sequential joint tests were computed as (true

positivetestA+true positivetestB)/(true positivetestA+false negativetestA) and (true negativetestB)/(true

negativetestB+false positivetestB) (Di Orio 1994) re-

spectively. The histological diagnosis on the surgical

specimens was regarded as the gold standard.

The aforementioned statistical parameters were

computed in the adenoma vs carcinoma groups and,

separately, in both oncocytic and non-oncocytic con-

ventional neoplasms.

The differences of each marker expression between

cytological and histological counterparts, adenomas

and carcinomas, and papillary and follicular carci-

nomas were analyzed by means of the c2 test, where

a value of P40.05 was considered as statistically sig-

nificant. Differences of marker expression between



oncocytic cell carcinomas and non-oncocytic carci-

nomas were also analyzed using two-tail Fisher exact

test, where a value of P40.01 was considered as

significant.

Results

Thyroid adenomas

Immunohistochemical analysis of FNAB cytological

specimens of adenomas with anti-GAL3 antibody

showed absent or scant (<10%) cytoplasmic expression

in the neoplastic cells of 47 out of 50 cases, whereas

nuclear staining was observed in focal areas or in

single cells of several adenoma specimens. Three

cases (6%) were diffusely positive in more than 80%

of neoplastic cells. Histologically, these cases were

proven adenomas associated with marked cytological

atypia and incomplete (non-full thickness) capsular

penetration. Such surgical specimens had the same

pattern of GAL3 staining. Normal follicular cells

surrounding each tumour nodule were GAL3 nega-

tive. Macrophages, endothelial cells and polymor-

phonuclear inflammatory cells expressed GAL3, as

expected.

HBME-1 was negative or had cell-membrane and

apical immunoreactivity in less than 10% of neo-

plastic cells in 48 out of 50 cytological cases. Two

samples (4%) had cell membrane and apical staining

of more than 80% of tumour cells. The same pattern

of cell block staining was observed in the correspond-

ing surgical specimens, which were confirmed to be

adenomas, according to currently accepted diagnostic

criteria. Normal follicular thyroid cells were negative

for HBME-1, as well as fibroblasts, endothelial cells,

histiocytes, lymphocytes and polymorphonuclear

inflammatory cells.

TPO immunoreactivity in more than 50% of

neoplastic cells was observed in 43 out of 50 cytological

cases (Fig. 1A). Seven adenomas (14%) had cytoplas-

mic expression in less than 40% of neoplastic cells. The

same pattern of positive-staining cell block cases

was observed in the corresponding surgical specimens

(Fig. 1B). Peri-tumoral thyroid gland was strongly

positive. Fibroblasts, endothelial cells, histiocytes,

lymphocytes and polymorphonuclear inflammatory

cells were unreactive.

CK19 showed absent or focal cytoplasmic ex-

pression (<10% of neoplastic cells) in 45 out of 50

cases. Five cases (10%) showed positive staining in

the cytoplasm of more than 70% of neoplastic cells.

The same pattern of cell block staining was observed

in the corresponding surgical specimens. The normal

gland surrounding each tumour specimen was either

negative or only focally positive. None of the non-

follicular cells had any immunoreactivity.

All but one FNAB cytological cases were negative

or focally stained (<10%) for KS. The remaining case

(2%) was positive in more than 70% of the cells with

both cell membrane and cytoplasmic patterns. The

same pattern of cell block staining was observed in

the corresponding surgical specimens. Normal thyroid

surrounding tumour nodules was negative or contained

occasionally positive cells. None of the non-follicular

cells had any immunoreactivity. The prevalences of

individual marker expression in the benign tumours

are summarized in Table 1.

Thyroid follicular carcinomas

Twenty-eight out of thirty-three (84.8%) FNAB

sections of follicular carcinoma had a cytoplasmic

GAL3 expression in more than 10% malignant cells,

whereas five samples were either negative or focally

(<10%) positive. All but one (a follicular carcinoma

with an insular component) corresponding surgical

specimens had the same pattern of staining.

HBME-1 had cell-membrane and apical staining of

more than 10% neoplastic cells in 21 out of 33 cases

(63.6%). Ten out of the twelve negative cell blocks

were oncocytic tumours, corresponding to 71% of

all oncocytic cell carcinomas of this series. All the

corresponding surgical cases showed the same pattern

of staining.

Twenty-three out of thirty-three (69.7%) cyto-

logical specimens of follicular carcinomas were either

TPO negative or had cytoplasmic TPO immuno-

reactivity in less than 50% of neoplastic cells

(Fig. 1C), whilst the remaining ten samples were

diffusely positive (>60% of cells). All the correspond-

ing surgical specimens showed the same pattern of

staining (Fig. 1D).

CK19 was positive in 21 out of 33 (63.6%) FNABs

of follicular carcinomas. All but two corresponding

surgical cases showed the same staining pattern. Two

CK19-positive surgical specimens where either nega-

tive or only focally (<8%) positive in their correspond-

ing FNAB material.

Only 21.2% (seven out of 33) of FNAB cytological

samples of follicular cancers had more than 10%

cells positive for KS (at both cell-membrane and cyto-

plasmic level) (Fig. 1E), whereas in the surgical

specimen group one-third (10/33) of cases were posi-

tive (Fig. 1F). The prevalences of individual marker

expression in follicular carcinomas are summarized

in Table 1.



Figure 1 Immunoperoxidase staining of immunohistochemical markers in thyroid tumors. TPO is expressed in a follicular

adenoma in both (A) the FNAB cytological cell block and (B) the corresponding surgical specimen. Conversely, in a follicular

carcinoma, TPO is negative in both (C) the cytological cell block and (D) the corresponding surgical specimen. KS is strongly

expressed in these cases of (E and F) follicular carcinoma and (G and H) follicular variant of papillary carcinoma in both (E and

G) cytological cell blocks and (F and H) the corresponding surgical specimens. Non-oncocytic carcinomas generally diffusely

express (I) GAL3 and (M) HBME-1 and (O) only focally CK19. Conversely, oncocytic follicular carcinomas are strongly positive

for (L) GAL3 and (P) CK19, rather than (N) HBME-1. (Immunoperoxidase, r160).



Thyroid papillary carcinomas

GAL3 had diffuse (over two-thirds of cancer cells)

cytoplasmic expression in 41 out of 42 (97.6%) FNAB

cytological cases. All the corresponding surgical speci-

mens were moderately to diffusely stained for GAL3.

Thirty-nine out of forty-two (92.9%) FNAB cyto-

logical samples had diffuse HBME-1 immunoreactivity

(>50% of malignant cells), whilst three cases were

either negative or only focally (<10%) positive. One

out of the three oncocytic papillary carcinomas was

negative. All corresponding surgical specimens showed

the same staining pattern.

With regard to TPO, 37 out of 42 (76.2%) FNAB

cytological specimens were either negative or stained

<50% of neoplastic cells. All the corresponding

surgical samples expressed TPO with the same pattern

of staining.

CK19 was positive in 36 out of 42 (85.7%) cases. Six

specimens were either negative or had focal (<10%)

immunoreactivity. All but one corresponding surgical

cases had the same pattern of staining.

KS was diffusely expressed in 29 out of 42 (69.0%)

FNAB cytological specimens (Fig. 1G), whereas the

other 13 were either negative or showed only occasional

positive cells. All but three corresponding surgical

samples showed the same pattern of staining (Fig. 1H).

The prevalences of individual marker expression in

papillary carcinomas are summarized in Table 1.

Statistical analysis

Single marker

No significant differences were observed between the

expression of each marker in FNAB cytological and

surgical specimens. All five markers were signifi-

cantly more expressed (GAL3, HBME-1, CK19, KS)

or reduced (TPO) in carcinomas than adenomas

(P<0.001). Moreover, GAL3, HBME-1, CK19 and

KS expression, as well as TPO immunoreactivity

reduction were significantly more associated with

papillary carcinomas than with follicular carcinomas

(P<0.05). In the papillary carcinoma group, no dif-

ferences were observed for all markers tested among

pure follicular variants and cases showing minor

classical or solid/trabecular growth patterns.

Although all markers were highly specific (o90%)

in both cytological and surgical samples, they notice-

ably differed in sensitivity, which ranged from 48%

for KS to 92% for GAL3 in cytological specimens

(Table 2), and from 56% for KS to 94% for GAL3

in the corresponding surgical specimens.

GAL3 and HBME-1 provided a higher accuracy

than other markers in both FNAB cytological and

surgical materials. In particular, GAL3 was less speci-

fic but more sensitive than HBME-1 (Table 2). A pro-

gressive decrease of accuracy values was found for

TPO, CK19 and KS (Table 2).

No significant differences were observed between

the expression of GAL3 (Fig. 1I and L), TPO, CK19

and KS in oncocytic cell tumours and non-oncocytic

ones on both cytological and surgical specimens.

Conversely, HBME-1 expression was significantly

more associated with non-oncocytic carcinomas than

oncocytic ones (P<0.001) in both FNAB cytological

(Fig. 1M and N) and surgical samples. HBME-1

showed a lower diagnostic accuracy in oncocytic

lesions (50%) than CK19 (Fig. 1O and P) and TPO

(Table 3).

Table 1 Prevalence of positivity of immunohistochemical markers in the benign and malignant thyroid tumour groups.

Cytological samples Surgical samples

GAL3 HBME-1 TPO CK19 KS GAL3 HBME-1 TPO CK19 KS

All tumours

Follicular adenoma 3/50 2/50 43/50 5/50 1/50 3/50 2/50 46/50 5/50 1/50

Follicular carcinomas 28/33 21/33 10/33 21/33 7/33 29/33 21/33 10/33 23/33 10/33

Papillary carcinomas* 41/42 39/42 5/42 36/42 29/42 42/42 39/42 5/42 37/42 32/42

Oncocytic subset of tumours




Non-oncocytic subset of tumours




*Histologically proven as pure follicular variants of papillary carcinomas or papillary carcinomas with a predominant follicular patternof growth.



Table 3 Discrimination between oncocytic and non-oncocytic thyroid FNAB indeterminate lesions by single marker or by

simultaneous/sequential combinations of markers.

Oncocytic Non-oncocytic

SN (%) SP (%) PV+ (%) PV- (%) AC (%) SN (%) SP (%) PV+ (%) PV- (%) AC (%)

Single marker

GAL3 88.00 100.00 100.00 77.78 91.67 93.10 93.02 94.74 90.91 93.07

CK19 64.71 100.00 100.00 53.85 75.00 79.31 88.37 90.20 76.00 83.17

TPO 64.71 85.71 91.67 50.00 70.83 84.48 86.05 89.09 80.43 85.15

HBME-1 35.29 85.71 85.71 35.29 50.00 93.10 97.67 98.18 91.30 95.05

KS 23.53 100.00 100.00 35.00 45.83 55.17 97.67 96.97 61.76 73.27

Two simultaneous markers

GAL3+CK19 100.00 100.00 100.00 100.00 100.00 98.28 81.40 87.69 97.22 91.09

GAL3+TPO 100.00 85.71 94.44 100.00 95.83 94.83 79.07 85.94 91.89 88.12

HBME-1+TPO 64.71 71.43 84.62 45.45 66.67 98.28 83.72 89.06 97.30 92.08

GAL3+HBME-1 94.12 85.71 94.12 85.71 91.67 98.28 90.70 93.44 97.50 95.05

Two sequential markers

GAL3+CK19* 100.00 100.00 – – – – – – – –

GAL3+TPO* 100.00 85.71 – – – 94.83 85.00 – – –

GAL3+HBME-1* – – – – – 98.28 97.50 – – –

HBME-1+GAL3* – – – – – 98.28 92.86 – – –

SN, sensitivity; SP, specificity; PV+, positive predictive value; PV - , negative predictive value; AC, accuracy; - , no computed.

*Marker of second instance. The highest results are shaded.

Table 2 Discrimination between benign and malignant thyroid lesions by single marker or by simultaneous/sequential

combinations of markers in cytological specimens.

SN (%) SP (%) PV+ (%) PV - (%) AC (%)

Single marker

GAL3 92.00 94.00 95.83 88.68 92.80

HBME-1 80.00 96.00 96.77 76.19 86.40

TPO 80.00 86.00 89.55 74.14 82.40

CK19 76.00 90.00 91.94 71.43 81.60

KS 48.00 98.00 97.30 55.68 68.00

Two simultaneous markers

GAL3+HBME-1 97.33 90.00 93.59 95.74 94.40

GAL3+CK19 98.67 84.00 90.24 97.67 92.80

HBME-1+CK19 92.00 88.00 92.00 88.00 90.40

GAL3+TPO 96.00 80.00 87.80 93.02 89.60

HBME-1+TPO 90.67 82.00 88.31 85.42 87.20

Three simultaneous markers

GAL3+HBME-1+CK19 100.00 82.00 78.57 100.00 92.60

GAL3+HBME-1+TPO 98.67 76.00 86.05 97.44 89.60

GAL3+TPO+CK19 98.67 70.00 83.15 97.22 87.20

Two sequential markers

GAL3+HBME-1* 97.33 95.74 – – –

GAL3+CK19* 98.67 89.36 – – –

GAL3+TPO* 96.00 85.11 – – –

SN, sensitivity; SP, specificity; PV+, positive predictive value; PV - , negative predictive value; AC, accuracy; – not computed.

*Marker of second instance. The highest results are shaded.



Simultaneous and sequential associations of markers

The highest diagnostic accuracy of the two-marker

simultaneous combinations in discriminating carci-

nomas from adenomas on FNAB cytological samples

was obtained by GAL3 plus HBME-1 (94.40%)

and GAL3 plus CK19 (92.80%). The simultaneous

association of GAL3 with HBME-1 showed a higher

specificity (90.00%) and a lower sensitivity (97.33%)

than that of GAL3 with CK19 (Table 2).

The best three-marker simultaneous combination

was GAL3, HBME-1 and CK19, which had a

sensitivity of 100%, but a relatively low specificity

(82.00%) (Table 2).

The sequential associations of HBME-1 or CK19 to

GAL3 showed a higher combined sensitivity (97.33%

and 98.67% respectively) and specificity (95.74% and

89.36% respectively) than the other sequential asso-

ciations evaluated (Table 2). GAL3/HBME-1 and

GAL3/CK19 sequential associations were superior

to the corresponding simultaneous combinations in

terms of specificity (95.74% and 89.36% vs 90.00%

and 84.00% respectively), and identical in terms of

sensitivity (Table 2).

On FNAB cytological specimens of oncocytic cell

neoplasms, the simultaneous combination of GAL3

with CK19 showed 100% of both sensitivity and speci-

ficity, strictly followed by that of GAL3 with TPO.

The same values of sensitivity and specificity were

obtained by a sequential coupling of the two markers

(Table 3).

In non-oncocytic cytological FNAB samples, the

highest values of sensitivity, specificity and accuracy

of the two-marker simultaneous combinations were

obtained by GAL3 with HBME-1 (98.28%, 90.70%

and 95.05% respectively) (Table 3). In non-oncocytic

tumours, the GAL3/HBME-1 sequential association

was superior to its simultaneous combination in terms

of specificity (97.50% vs 90.70%) and identical in

terms of sensitivity (Table 3).

Discussion

In this study, the expression of selected markers

(GAL3, HBME-1, TPO, CK19 and KS) was assessed

in a relatively large series of FNAB cytological and

surgical thyroid samples using commercial species-

specific monoclonal antibodies and a biotin-free detec-

tion system, in order to avoid interfering technical

factors (e.g. aspecific antigen cross-reactivity of non-

species-specific antibodies and endogenous biotin-like

activity) (Herrmann et al. 2002, Bartolazzi et al. 2003,

Saggiorato et al. 2004) which may be responsible for

most of the reported discrepancies, particularly in the

cases of GAL3 and HBME-1 (Nascimento et al. 2001,

Mai et al. 2002, Mehrotra et al. 2004).

A first matter was the selection of markers. Accord-

ing to the reports in the literature, GAL3, HBME-1

and CK19 proved the most reliable ones, due to their

reproducibility and also to the availability of commer-

cial antibodies. All three selected markers (GAL3,

HBME-1 and CK19) were claimed to be useful for

both follicular and papillary carcinomas, although

the latter are generally more strongly reactive for

such molecules. To expand the search for an optimal

panel of markers, we also introduced two novel anti-

bodies, claimed to be able to distinguish benign from

malignant differentiated follicular tumours: anti-TPO,

which was actually tested more than 10 years ago

(De Micco et al. 1991), but became commercially avail-

able only recently, and anti-KS, which is reported

mainly expressed in papillary carcinomas.

A second important point was to set cut-off values

for each marker, being their expression not subjected

to the all-or-none law. We have thus defined the

most appropriate cut-off in an attempt to reduce false

negative results (that means to ensure thyroidectomy

to all patients with carcinomas) as much as possible,

to the detriment of a slight increase of false positive

diagnoses. According to the aforementioned criteria,

the best compromise between sensitivity and specificity

was reached setting at 10% the cut-off value for

GAL3, HBME-1, CK19 and KS expression, and at

50% that for TPO. In particular, the cut-off values

(HBME-1, CK19 and TPO) here assigned were slightly

more restrictive than those reported by some other

authors (vanHoeven et al. 1998, Christensen et al. 2000,

Raffaelli et al. 2001, Khurana et al. 2003). We suggest

that these discrepancies may depend on the use of a

biotin-free detection system, which is potentially able

to reduce false stainings.

Based on the selected cut-offs for each markers,

it was clear from our series that GAL3 was superior

to other markers in identifying the majority of malig-

nant tumours.

HBME-1 and CK19 were here confirmed to be

valuable markers, while the novel KS was acceptable

for papillary carcinoma cases only. Intact TPO was

under-expressed in 80% of malignant cases, but its

association with GAL3 did not considerably improve

the diagnostic accuracy.

The results of our statistical analysis have indicated

that the use of three or more markers at a time does

not increase the FNAB diagnostic accuracy up to

100%, and is thus useless, especially at a time of money

restrictions and scant resources. In practical terms,



we observed that just two markers (one being GAL3

and the other HBME-1 or CK19) are able to provide a

good diagnostic accuracy (>90%) with a sensitivity as

high as 97% and a specificity of 90%.

The last point was how to plan the immuno-

histochemical panel analysis, keeping in mind the

cost-benefit problems and the turn-around time of

pathology reports. Hence, we wanted to check whether

there was any benefit from the simultaneous (two

or more markers simultaneously tested in 1 day) or

sequential (one marker first, followed by a second

marker in the case of a first marker-negative result

only) analysis of the selected molecules.

We have here shown that sequential analysis of

GAL3 and HBME-1 or GAL3 and CK19 gave the

same sensitivity (approximately 97%) as their simul-

taneous combinations, and a higher specificity

(approximately 90%) than the simultaneous one,

reducing the costs due the immunohistochemical

procedures. Finally, we compared the accuracy of the

different marker combinations in identifying malignant

tumours of the oncocytic type vs the conventional

type. Interestingly, we observed a marked difference

in the ability to recognize oncocytic carcinomas when

GAL3 was sequentially associated with CK19 (reach-

ing 100% of both sensitivity and specificity), rather

than HBME-1. Conversely, the latter proved more

valuable in identifying follicular (and papillary)

carcinomas of non-oncocytic types (98.28 of sensitivity

and 97.50 of specificity). This finding is in agreement

with the data in the literature on the reduced

expression of HBME-1 by oxyphilic thyroid tumours

(Mai et al. 2002, Volante et al. 2004a), and indicates

that the selection of immunohistochemical markers

for FNABs should be driven by the morphological

features of cytological material (e.g. HBME-1 should

be avoided in the presence of mitochondrion-rich

oncocytic cells, as its sensitivity for such malignant

tumours is as low as 35%).

Taken together, our results have provided useful

suggestions for the diagnostic management of patients

bearing thyroid nodules with an indeterminate FNAB

result. In our opinion, patients with FNAB cytology

of ‘follicular neoplasm’ and a positive GAL3 cyto-

logical test should be referred directly to the surgeon,

without any other immunohistochemical evaluation

and regardless of the clinical and ultrasound features.

In the case of a negative GAL3 test, we propose

the evaluation by a second marker represented by

HBME-1 for the non-oncocytic follicular neoplasms

or CK19 for the oncocytic cell tumours. The negative

result of the second marker (HBME-1 or CK19) should

encourage the clinician to use a follow-up programme,

including a further FNAB 6–12 months later (possibly

with immunohistochemical re-evaluation), and refer-

ring the patient to the surgeon when an increase in the

nodule size or a positive result in a sequential test is

observed. At worst, a diagnosis of thyroid malignancy

will be just slightly postponed. Conversely, in the case

of a positive result with the second marker the patient

should be referred to the surgeon for thyroidectomy

(Fig. 2). Needless to say patients with an indeterminate

FOLLOW-UP

Indeterminate

SURGERYY

Benign Malignant

HBME-1

NON-ONCOCYTICONCOCYTIC

GAL3 +

-

FNAB

CK19

+

+

-

-

Figure 2 Proposal of practical management of an indeterminate thyroid FNAB result (‘follicular neoplasm’), based on two

markers sequentially combined (GAL3+CK19 or GAL3+HBME-1).



FNAB result, but with clinical signs strongly sugges-

tive of malignancy (e.g. hard and fixed thyroid nodule,

lymphadenopathies, ultrasonographic features sugges-

tive of malignancy) should be treated accordingly and

referred to the surgeon, irrespective of the FNAB

cytology results.

Hence, we propose a diagnostic algorithm, of low

cost and easy interpretation, able to optimize the

management of patients with thyroid nodular disease,

reducing the number of unnecessary thyroidectomies

for benign neoplasms.

Acknowledgements

We wish to thank Dr Carlotta Sacerdote (Unit of

Cancer Epidemiology, University of Turin) for her

invaluable help in the statistical analysis of these

data, Dr Nicoletta Bergero for technical assistance and

Dr Armando Bartolazzi (Department of Pathology,

Saint Andrea Hospital, Rome) for helpful discussion.

Funding

This research was supported by grants from the

Regione Piemonte (D.D. n.63 of May 27 2002,

D.G.R. n.69-8040 of December 16 2002, D.D. n.104

of July 21 2003 and D.D. n.173 of October 30 2003),

Turin, and from the Italian Ministry of University and

Research, Rome, and Compagnia San Paolo, Progetto

Speciale Oncologia, Turin, Italy. RDP is a recipient

of a research fellowship from the Fondazione Inter-

nazionale di Ricerca in Medicina Sperimentale, Turin,

Italy. The authors declare that there is no conflict of

interest that would prejudice the impartiality of this

scientific work.

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