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© Urban &Fischer Verlag

Is ITF1 a Good Immunohistochemical Marker to

Distinguish Primary from Metastatic Lung

Jorge S. Reis-Filho1, Carla Carrilho 2, Carla Valenti 1, Dina Leitao 1,

Carlos A. Ribeiro 3 , Silvana G. A. Ribeiro 3 and Fernando C. Schmitt 1

'Institute of Pathology and Molecular Immunology at Porto University, IPATIMUP, Porto,

'Department of Pathology, Medical Faculty of Eduardo Mondlane University, Maputo,
JDepartment of Pathology, Medical Faculty of Minas Gerais, Brazil

Summary Introduction

To evaluate thc immunohistochcmical expression of Thyroid transcription factor 1 (TIFI) is a home-

thyroid transcription factor I (TTFI) in primary and odomain-containing nuclear transcription protein of the
metastatic pulmonary adenocarcinomas, and test the Nkx2 gene family that is normally expressed in certain
diagnostic accuracy of this antibody, two surgical areas of the central nervous system during embryogene-
pathologists independently evaluated 34 caseS of ade- sis {l, 2, 3/. TTFI expression is also well documented
nocarcinomas in the lung without clinical data and tried in tbe thyroid and lung 11, 2, 3}. In these tissues, TIF 1
to distinguish between primary and mctastatic cases activates the transcription of thyroglobulin and thy-
using histological criteria exclusively. Thirteen cases roperoxidase in the thyroid and the transcription of the
were primary in the lung and 21 were metastases of ex- surfactant B protein gene in lung epithelial cells [2/. In
trapulmonary adenocarcinomas: 6 from the endometri- fact, some authors {l, 2, 3/ have shown that Clara cells
um, 4 from the ovary, 3from the colon, 2 from the kid- and type II pneumocytes present high levels of this pro-
ney, 2 from the breast, 2 from the liver and 1 from the tein; these have been confirmed by immunohistochemi-
prostate. Afterward, the immunoreactivity of TTFI in cal {l, 2J and RNAse protection assays {3j.
these neoplasms was evaluated and correlated with Recently, TIFl has received great attention among
morphological and clinical data. The two pathologists surgical pathologists as a good immunohistochemical
were able to diagnose only 5 out of 13 cases of primary marker for lung and thyroid carcinomas {l, 2}. In differ-
lung adenocarcinomas (sensitivity of 38.46%) and also ent types of lung carcinomas, only adenocarcinomas,
misdiagnosed two primary malignancies as metastases. large cell carcinomas and small cell carcinomas are posi-
After correlation with TIFI data, the sensitivity in- tive for this protein fl, 2, 3, 4, 5, 6}. Antibodies against
creased to 61.53%. The specificity ofTIFI was 100%. this transcription factor have been widely tested as mark-
In conclusion, TIFI is a highly specific marker for pri- ers to disti nguish metastatic small cell carcinomas from
mary lung adenocarcinomas, and should be included in Merkel cell carcinomas {7/, as well as to distinguish lung
a panel of antibodies for the differential diagnosis be- adenocarcinomas from mesotheliomas [2. 8. 9].
tween primary and metastatic adenocarcinomas of the
lung. Address for correspondence: Fernando C. Schmitt, IPA-
TIMUP - Instituto de Pathoiogia e Imunologia Molecular da
Key words: Thyroid transcription factor - Lung cancer Universidadc do Porto, R. Roberto Frias, SIN. 4200 Porto,
- Adenocarcinoma - Immunohistochemistry Portugal.

Pathol. Res. Pract. 196: 83S-B40 (2000) 0344·033812000/ 196112-835 $15.0010

Table I. Clinical. histological, and immunohistochemical profile of the adenocarcinomas evaluated in this study 00
Case Primary site Morphological diagnosis Histological features TIFl TIFI Diagnosis considering TIFI !-
(tumor) Lung
I Breast Metastatic adenocarcinoma Poorly differentiated adenocarcinoma with solid areas + Metastatic adenocarcinoma <;;>
2 Breast Adenocarcinoma* Carcinoma with solid areas + Adenocarcinoma'" :!1
3 Colon Metastatic adenocarcinoma Tubular adenocarcinoma + Metastatic adenocarcinoma 5'
4 Colon Metastatic adenocarcinoma Mucinous adenocarcinoma + Metastatic adenocarcinoma :!.
5 Colon Metastatic adenocarcinoma Mucinous adenocarcinoma + Metastatic adenocarcinoma ~
6 Endometrium Metastatic adenocarcinoma Poorly differentiated adenocarcinoma with solid areas + Metastatic cndometrioid
7 Endometrium Metastatic adenocarcinoma Adenocarcinoma with mucinous foci + Metastatic adenocarcinoma
8 Endomctrium Metastatic adenocarcinoma Poorly differentiated adenocarcinoma with squamous foci + Metastatic endometrioid
9 Endometrium Metastatic adenocarcinoma Cribriform adenocarcinoma + Metastatic adenocarcinoma
10 Endometrium Metastalic adenocarcinoma Endometrioid adenocarcinoma + Metastatic endometrioid
II Endometrium Metastatic adenocarcinoma Endometrioid adenocarcinoma + Metastatic adenocarcinoma
12 Kidney Metastatic clear cell carcinoma Clear cell carcinoma + Metastatic clear cell carcinoma
13 Kidney Metastatic clear cell carcinoma Clear cell carcinoma + Metastatic clear cell carcinoma
14 Liver Metastatic adenocarcinoma Carcinoma with solid areas + Metastatic adenocarcinoma
15 Liver Metastatic adenocarcinoma Carcinoma with solid areas + Metastatic adenocarcinoma
16 Ovary Adenocarcinoma* Poorly differentiated adenocarcinoma with mucinous foci + Adenocarcinoma*
17 Ovary Adenocarcinoma* Poorly differentialed adenocarcinoma + Adenocarcinoma*
18 Ovary Metastatic adenocarcinoma Mucinous adenocarcinoma + Metastatic adenocarcinoma
19 Ovary Metastatic adenocarcinoma Moderately differentiated adenocarcinoma + Metastatic adenocarcinoma
20 Ovary Metastatic adenocarcinoma Mucinous adenocarcinoma + Metastatic adenocarcinoma
21 Prostate Metastatic adenocarcinoma Adenocarcinoma with cribriform and solid areas + Metastatic adenocarcinoma
22 Lung Adenocarcinoma* Solid carcinomas with mucus formation + Adenocarcinoma*
23 Lung Metastatic carcinoma Solid carcinomas with mucus formation + Metastatic clear cell carcinoma
24 Lung Adenocarcinoma* Solid carcinomas with mucus formation + Adenocarcinoma*
25 Lung Metastatic adenocarcinoma Solid carcinomas with mucus formation + Metastatic adenocarcinoma
26 Lung Adenocarcinoma* Solid carcinomas with mucus formation + Adenocarcinoma*
27 Lung Primary Adenocarcinoma Acinar adenocarcinoma with focal squamous differentiation + + Primary Adenocarcinoma
28 Lung Adenocarcinoma* Acinar adenocarcinoma + + Primary Adenocarcinoma
29 Lung Primary Adenocarcinoma Acinar adenocarcinoma + + Primary Adenocarcinoma
30 Lung Adenocarcinoma* Solid carcinomas with mucus formation + + Primary Adenocarcinoma
31 Lung Primary Adenocarcinoma Acinar adenocarcinoma + + Primary Adenocarcinoma
32 Lung Primary Adenocarcinoma Bronchioloal veolar carcinoma + + Primary Adenocarcinoma
33 Lung Metastatic adenocarcinoma Acinar adenocarcinoma + + Primary Adenocarcinoma
34 Lung Primary Adenocarcinoma Papillary adenocarcinoma + + Primary Adenocarcinoma

*: cases in which a diagnosis of primary or metastatic adenocarcinoma could not be achieved based on morphological data
TIF-I in Lung Adenocarcinomas ' 837

The differential diagnosis of primary and metastatic colon, 2 from the kidney, 2 from the breast, 2 from the
lung adenocarcinomas presents a problem in routine liver, and I from the prostate.
surgical pathology, mainly when there is not a con- The morphological diagnoses of the cases are sum-
firmed primary site {l, 5, 6, 10]. In this paper, the au- marized in Table 1. Using only the morphological crite-
thors evaluated the role of TIFI as an ancillary tech- ria,S cases were diagnosed as primary adenocarcino-
nique in the differentiation of primary and metastatic mas, 22 as metastatic adenocarcinomas, and in 7 cases
lung adenocarcinomas, testing its sensitivity and speci- it was not possible to determine, based on morphologi-
ficity, as well as describing the improvement in the di- cal criteria, if the tumor was primary or metastatic,
agnosis of primary lung adenocarcinomas,
Immunohistochemical analysis

Methods TTFI was positive in 8 cases (Fig, I), and all of these
cases were primary lung adenocarcinomas (Table 2).
Two surgical pathologists, without any clinical data, inde- According to the WHO's classification, these cases cor-
pendently evaluated 34 cases of adenocarcinomas in the lung, responded to 4 pure acinar adenocarcinomas, one acinar
In this fim analysis, the pathologists tried to diagnose the ade- adenocarcinoma with focal squamous differentiation,
nocarcinomas as primary or metastatic using only morpholog- one solid carcinoma with mucus formation, one bron-
ical criteria [11].
chioloalveolar carcinoma, and one papillary adenocar-
A second step was to evaluate the immunocxpression of
TIFI in the neoplastic cells and in the normal lung, trying to cinoma, Five primary lung adenocarcinomas were neg-
confirm the diagnosis or primary lung carcinomas. ative, and none of the metastatic cases presented any
The immunohistochemical assays were performed accord- positivity for this antibody, The negative primary lung
ing to the avidin-biotin-peroxidase technique as previously
describcd [12/, with monoclonal antibody raised against thy-
roid transcription factor-I (8070311 culture supernatant -
Neomarkers, Union City, CAl, To exclude equivocal reac-
tions, an at least moderate staining intensity in more than 10%
of the tumor cells was required as a relevant positive reaction
'I]. Only nuctear staining was considered as positive,
After that, the two surgical pathologists and a third pathol-
ogist subclassified the primary lung adenocarcinomas accord-
ing to the WHO's classification [13].
The sensitivity and the specificity of the immunostaining
were defined as the following:
True positives
Sensitivity = - --,-,--'--,::-c----,--
True posiLives + False negatives

True negatives
True negatives + False positives
Fig, I, Photomicrography of a primary lung adenocarcinoma
The authors also evaluated the number of cases in which showing nuclear positivity for TIFI. (Avidin-biotin-peroxi-
TIFI had changed the diagnosis, improving the definition of dase/Diaminobenzidine x400)
primary or metastatic lung adenocarcinomas.
In the present study, a definitive classification of the adeno-
carcinomas as primary or metastatic was provided according Table 2. TIFl immunoreactivity in primary and metastatic
to clinical data, radiologicaJ features, patient'S follow-up and
lung adenocarcinomas
in some cases by autopsy, The cases were considered as prima-
ry lung tumors when no other primary site had been found, TIF immunoreactivity

Cases Positive Negative Total

Primary lung adenocarcinoma 8 5 13
Evaluation of the morphological data Metastatic adenocarcinomas 0 21 21
The cases evaluated in this study included 13 prima- Total 8 26 34
ry lung adenocarcinomas, and 21 metastatic adenocar-
cinomas, Among the metastases, the primary sites were: Sensitivity = 61.53%
6 from the endometrium, 5 from the ovary, 3 from the Specificity = 100%
838 . J. S. Reis-Filho et al.

Table 3. Histological and immunohistochemical features of not possible by morphology, 2were positive and 6 were
the primary lung adenocarcinomas negative for TTFI. One of the 22 cases diagnosed mor-
phologically as metastasic was positive for TIFI.
Histological diagnosis TIFI
Compari50n of the hi5tological and immunohi5tochemi-
Solid carcinomas with mucus formation cal finding5 with the clinical data
Solid carcinomas with mucus formation
Solid carcinomas with mucus formation The clinical, pathological, and immunohistochemical
Solid carcinomas with mucus formation profile of all cases is described in Table I.
SaUd carcinomas with mucus formation The sensitivity of the surgical pathologists without
Acinar adenocarcinoma with focal squamous + any clinical or immunohistochemical data to diagnose
differentiation primary lung adenocarcinomas was 38.46% (5 out of 13
Acinar adcnocan.:inorna + cases). Considering TTFI results, 3 additional cases
Acinar adenocarcinoma + could be included among the primary cancers, leading
Solid carcinomas with mucus formation + to a sensitivity of 61.53%.
Acinar adenocarcinoma + In one case misdiagnosed as a metastasis and in 2
Bronchioloalveolar carcinoma +
Acinar adenocarcinoma +
cases without a definition of primary or metastatic,
Papillary adenocarcinoma + TTFI immunostaining was positive, changing the diag-
noses to primary lung adenocarcinomas. After this cor-
relation, 8 cases were considered primary malignancies,
and 20 metastases. In 6 cases in which the distinction of
primary or metastases could not be achieved, TTF I did
not contribute further to the diagnosis.
Compared to the clinical data, all 8 cases defined as
primary adenocarcinomas were correctly diagnosed.
Eighteen out of the 20 cases diagnosed as metastases
after TTFI evaluation were correctly diagnosed; 2 cases
were primary adenocarcinomas misinterpreted as
metastases. Considering the 6 adenocarcinomas in
which the diagnosis of primary or metastatic adenocar-
cinomas could not be achieved by morphology and
were negative for TTFI, 3 were metastatic and 3 were
primary lung adenocarcinomas. In all these cases, the
internal control was positive.

Fig. 2. Normal lung alveolar epithelial cells showing nuclear
positivity for TIFt (arrows) . In the center, nests of cells from Primary lung adenocarcinomas are one of the most
a metastatic adenocarcinoma without nuclear staining.
prevalent causes of metastatic disease with an unknown
(Avidin-biotin-peroxidase/Diaminobenzidine x400)
primary site; furthermore, the lungs are frequent targets
for metastatic carcinomas, coming mainly from the kid-
ney, breast, prostate, and liver, among others [1,3,5,6,
adenocarcinomas were all solid carcinomas with mucus 9, II/. The distinction hetween primary and secondary
formation (Table 3). In all cases, the adjacent lung tis- adenocarcinomas affecting the lungs is a difficult task,
sue presented immunoreactivity for TTFI in the nuclei especially when we have a small biopsy or fine needle
of type II pneumocytes and Clara cells (Fig. 2). aspiration material.
Recently, several reports have been published con-
cerning new immunohistochemical antibodies against
Evaluation of the diagno5tic accuracy with and without
proteins and tissue-specific transcription factors as
markers to distinguish primary from metastatic lung
When the pathologists correlated the TTF I im- adenocarcinomas [l, 5, 6, 9, 10]. TTFI is considered
munostaining with the morphological data, the five one of the best markers to distinguish primary from
cases diagnosed as primary lung adenocarcinomas were metastatic primary lung adenocarcinomas II , 5, 6, 10};
confirmed. Among the 8 cases in which differentiation besides, it is commercially available and able to be em-
between metastatic and primary adenocarcinoma was ployed in paraffin-embedded tissue specimens [9].
TTF-I in Lung Adenocarcinomas 839

Table 4. TTF1 immunoreactivity in primary and metastatic lung adenocarcinomas in different reported series

Authors (Reference) Primary pulmonary tumors Extrapuhnonary tumors Sensitivity (%) Specificity (%)
(# of adenocarcinomas)

Kaufmann et al. [l] 138 (98) 276 75 98

Khoor et al. [2/ 370 (208) 95 76 100
Fabbro et al. [3 J* 43 (15) 27
Bejarano et al. [4/ 57 177 76 99
Harlamert et al. [51 41 (21) 6 76 100
Di Loreto et al. [61 33 (33) 24 57.5 100
Di Loreto et al. [81 17 (8) 62.5
Ordonez NG {9J 40 (40) 145
Borrinski et al. [10/ 18 (18) 12 67 100
Present study 13 (13) 21 61.5 100

*: studies in which only primary lung tumors were evaluated

The sensitivity of TTFI in lung adenocarcinomas in even primary lung adenocarcinomas presenting mucin
the different reported series ranged from 57.5 % to 76% production arc usually negative for TTFI. Taking this
(Table 4) [I, 2, 3, 4, 5, 6, 8,9, 1Oj. Excluding thyroid aspect into account, we suggest that TTFI be incorpo-
neoplasms, the TTFI specificity ranged from 99% {4/ rated in a panel for the differential diagnosis of adeno-
to 100% for primary lung carcinomas [1 , 2, 3, 5, 6, 8, carcinomas with an unknown primary site, mainly in
9]. In our series, the sensitivity was 61.53% and the small specimens and in fine needle aspiration biopsies.
specificity 100%. Five cases of primary lung carcino-
mas werc negative for TTFI; in all these cases, the au-
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