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PATHOlOGY

RESEARCH AND PRAcnCE

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&Fischer Verlag httpllwww.urbanfischer.defjournalslprp Is ITF1 a Good Immunohistochemical Marker to Distinguish

Is ITF1 a Good Immunohistochemical Marker to Distinguish Primary from Metastatic Lung Adenocarcinomas?

Jorge S. Reis-Filho 1, 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, Portugal; 'Department of Pathology, Medical Faculty of Eduardo Mondlane University, Maputo, Mozambique; JDepartment of Pathology, Medical Faculty of Minas Gerais, Brazil

Summary

To evaluate thc immunohistochcmical expression of thyroid transcription factor I (TTFI) in primary and metastatic pulmonary adenocarcinomas, and test the diagnostic accuracy of this antibody, two surgical pathologists independently evaluated 34 caseS of ade- nocarcinomas in the lung without clinical data and tried to distinguish between primary and mctastatic cases using histological criteria exclusively. Thirteen cases were primary in the lung and 21 were metastases of ex- trapulmonary adenocarcinomas: 6 from the endometri- um, 4 from the ovary, 3from the colon, 2 from the kid- ney, 2 from the breast, 2 from the liver and 1 from the prostate. Afterward, the immunoreactivity of TTFI in these neoplasms was evaluated and correlated with morphological and clinical data. The two pathologists were able to diagnose only 5 out of 13 cases of primary lung adenocarcinomas (sensitivity of 38.46%) and also misdiagnosed two primary malignancies as metastases. After correlation with TIFI data, the sensitivity in- creased to 61.53%. The specificity ofTIFI was 100%.

In conclusion, TIFI is a highly specific marker for pri- mary lung adenocarcinomas, and should be included in

a panel of antibodies for the differential diagnosis be- tween primary and metastatic adenocarcinomas of the lung.

Key words: Thyroid transcription factor - Lung cancer

- Adenocarcinoma - Immunohistochemistry

Pathol. Res. Pract. 196: 83S-B40 (2000)

Introduction

Thyroid transcription factor 1 (TIFI) is a home- odomain-containing nuclear transcription protein of the Nkx2 gene family that is normally expressed in certain areas of the central nervous system during embryogene- sis {l, 2, 3/. TTFI expression is also well documented in tbe thyroid and lung 11, 2, 3}. In these tissues, TIF 1 activates the transcription of thyroglobulin and thy- roperoxidase in the thyroid and the transcription of the surfactant B protein gene in lung epithelial cells [2/. In fact, some authors {l, 2, 3/ have shown that Clara cells and type II pneumocytes present high levels of this pro- tein; these have been confirmed by immunohistochemi- cal {l, 2J and RNAse protection assays {3j. Recently, TIFl has received great attention among surgical pathologists as a good immunohistochemical marker for lung and thyroid carcinomas {l, 2}. In differ- ent types of lung carcinomas, only adenocarcinomas, large cell carcinomas and small cell carcinomas are posi- tive for this protein fl, 2, 3, 4, 5, 6}. Antibodies against this transcription factor have been widely tested as mark-

small cell carcinomas from

Merkel cell carcinomas {7/, as well as to distinguish lung adenocarcinomas from mesotheliomas [2. 8. 9].

ers to disti nguish metastatic

Address for correspondence: Fernando C. Schmitt, IPA-

TIMUP - Instituto de Pathoiogia e Imunologia Molecular da Universidadc do Porto , R. Roberto Frias, SIN. 4200 Porto, Portugal.

0344·0 33812000/ 19 6112-835 $15.0010

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Metastatic cndometrioid adenocarcinoma Metastatic adenocarcinoma Metastatic endometrioid adenocarcinoma Metastatic adenocarcinoma Metastatic endometrioid adenocarcinoma Metastatic adenocarcinoma+ Metastatic clear cell carcinoma+

Metastatic clear cell carcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Adenocarcinoma* Adenocarcinoma* Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma Metastatic adenocarcinoma+ Adenocarcinoma* Metastatic clear cell carcinoma Adenocarcinoma* Metastatic adenocarcinoma Adenocarcinoma* Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma Primary Adenocarcinoma

TIFICase

adenocarcinomaI

adenocarcinoma+

Metastatic adenocarcinoma

adenocarcinoma3

Metastatic considering

'"Adenocarcinoma+

Metastatic

Metastatic

Diagnosis

+ Lung

TIFI

+

+

+

++

+

+

+

+

+

+

++

+

+

+

+

+ +

+

+

+

+

+

+

+

+

+

+

+

(tumor)

TIFl

*: cases in which a diagnosi s of primary or metastatic adenocarcinoma could not be achieved based on morphological data

+

+

+

+

+

+

Solid carcinomas with mucus formation Acinar adenocarcinoma with focal squamous differentiation

Poorly differentiated adenocarcinoma with squamous foci8

Clear cell carcinoma Carcinoma with solid areas Carcinoma with solid areas Poorly differentiated adenocarcinoma with mucinou s foci

features adenocarcinoma with solid areasBreast

Poorly differentiated adenocarcinoma with solid areas6

Clinical. histological, and immunohistochemical profile of the adenocarcinomas evaluated in this study

Poorly differentialed adenocarcinoma Muc inou s adenocarcinoma Moderately differentiated adenocarcinoma Mucinous adenocarcinoma Adenocarcinoma with cribriform and solid areas Solid carcinomas with mucus formation Solid carcinomas with mucu s formation Solid carcinomas with mucus formation

Solid carcinomas with mucu s formation30

Solid carcinomas with mucu s formation25

Adenocarcinoma with mucinous foci

Endometrioid adenocarcinoma10

Endometrioid adenocarcinoma

Acinar adenocarcinoma Bronchioloal veolar carcinoma Acinar adenocarcinoma Papillary adenocarcinoma

Cribriform adenocarcinoma9

with solid areas2

adenocarcinoma4

adenocarcinoma5

adenocarcinoma

Acinar adenocarcinoma Acinar adenocarcinoma

Clear cell carcinoma12

Poorly differentiated

Histological

Carcinoma

Mucinous

Mucinous

Tubular

Metastatic clear cell carcinoma

Metastatic clear cell carcinoma

Metastatic adenocarcinoma

Metastatic adenocarcinoma

Metastatic adenocarcinoma

Metastatic adenocarcinoma33

Metastatic adenocarcinoma20

Metastatic adenocarcinoma

Metastalic adenocarcinoma

Metastatic adenocarcinomaII

Metastatic adenocarcinoma

Metastatic adenocarcinoma

adenocarcinoma

Adenocarcinoma * Metastatic adenocarcinoma

Metastatic adenocarcinoma Adenocarcinoma* Metastatic carcinoma

Metastatic adenocarcinoma

Metastatic ade nocarcinoma Metas tatic a denocarcinoma

adenocarcinoma

adenocarcinoma

Adenocarcinoma * Primary Adenocarcinoma Adenocarcinoma * Primary Adenocarcinoma

Primary Adenocarcinoma Primary Adenocarcinoma

Primary Adenocarcinoma34

diagnosis

Adenocarcinoma *24

Adenocarcinoma*16

Adenocarcinoma*

Adenocarcinoma*

Morphological

Metastatic

Metastatic

Metastatic

Endometrium

Endometrium

Endomctrium

Endometrium

Endometrium7

Endometrium

Primary site

Prostate

Kidney

Kidney

Breast

Ovary

Ovary

Ovary

Ovary

Ovary

Colon

Colon

Colon

Liver

Liver15

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Lung

Table I.

26

28
29

31
32

21
22

18
19

13
14

23

17

27

The differential diagnosis of primary and metastatic lung adenocarcinomas presents a problem in routine surgical pathology, mainly when there is not a con- firmed primary site {l, 5, 6, 10]. In this paper, the au- thors evaluated the role of TIFI as an ancillary tech- nique in the differentiation of primary and metastatic lung adenocarcinomas, testing its sensitivity and speci- ficity, as well as describing the improvement in the di- agnosis of primary lung adenocarcinomas,

Methods

Two surgical pathologists, without any clinical data, inde-

pendently evaluated 34 cases of adenocarcinomas in the lung, In this fim analysis, the pathologists tried to diagnose the ade- nocarcinomas as primary or metastatic using only morpholog- ical criteria [11].

A second step was to evaluate the immunocxpression of

TIFI in the neoplastic cells and in the normal lung, trying to

confirm the diagnosis or primary

The immunohistochemical assays were performed accord-

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-

lung carcinomas.

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

Specificity

True negatives

True negatives + False positives

The authors also evaluated the number of cases in which

TIFI had changed the diagnosis, improving the definition of

primary or metastatic lung adenocarcinomas. In the present study, a definitive classification of the adeno- carcinomas as primary or metastatic was provided according to clinical data, radiologicaJ features, patient'S follow-up and

in some cases by autopsy, The cases were considered as prima- ry lung tumors when no other primary site had been found,

Results

Evaluation of the morphological data

The cases evaluated in this study included 13 prima- ry lung adenocarcinomas, and 21 metastatic adenocar- cinomas, Among the metastases, the primary sites were:

6 from the endometrium, 5from the ovary, 3 from the

TIF-I in Lung Adenocarcinomas ' 837

colon, 2 from the kidney, 2 from the breast, 2 from the liver, and I from the prostate. The morphological diagnoses of the cases are sum- marized in Table 1. Using only the morphological crite- ria,S cases were diagnosed as primary adenocarcino- mas, 22 as metastatic adenocarcinomas, and in 7 cases it was not possible to determine, based on morphologi- cal criteria, if the tumor was primary or metastatic,

Immunohistochemical analysis

TTFI was positive in 8 cases (Fig, I), and all of these cases were primary lung adenocarcinomas (Table 2). According to the WHO's classification, these cases cor- responded to 4 pure acinar adenocarcinomas, one acinar adenocarcinoma with focal squamous differentiation, one solid carcinoma with mucus formation, one bron- chioloalveolar carcinoma, and one papillary adenocar- cinoma, Five primary lung adenocarcinomas were neg- ative, and none of the metastatic cases presented any positivity for this antibody, The negative primary lung

any positivity for this antibody, The negative primary lung Fig, I, Photomicrography of a primary lung

Fig, I, Photomicrography of a primary lung adenocarcinoma showing nuclear positivity for TIFI. (Avidin-biotin-peroxi- dase/Diaminobenzidine x400)

Table 2. TIFl immunoreactivity in primary and metastatic lung adenocarcinomas

TIF immunoreactivity

Cases

Positive

Negative

Total

Primary lung adenocarcinoma

8

5

13

Metastatic adenocarcinomas

0

21

21

Total

8

26

34

Sensitivity = 61.53% Specificity = 100%

838 . J. S. Reis-Filho et al.

Table 3. Histological and immunohistochemical features of

the primary lung adenocarcinomas

Histological diagnosis

TIFI

(tumour)

not possible by morphology, 2were positive and 6 were negative for TTFI. One of the 22 cases diagnosed mor- phologically as metastasic was positive for TIFI.

Compari50n of the hi5tological and immunohi5tochemi- cal finding5 with the clinical data

Solid carcinomas with mucus formation Solid carcinomas with mucus formation Solid carcinomas with mucus formation Solid carcinomas with mucus formation

SaUd carcinomas with mucus formation Acinar adenocarcinoma with focal squamous

The clinical, pathological, and immunohistochemical profile of all cases is described in Table I. The sensitivity of the surgical pathologists without

+ any clinical or immunohistochemical data to diagnose primary lung adenocarcinomas was 38.46% (5 out of 13

+ cases). Considering TTFI results, 3 additional cases

+ could be included among the primary cancers, leading

+ to a sensitivity of 61.53%.

+ In one case misdiagnosed as a metastasis and in 2

+ + cases without a definition of primary or metastatic,

+ 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

differentiation

Acinar adcnocan.:inorna Acinar adenocarcinoma

Solid carcinomas with mucus formation

Acinar adenocarcinoma

Bronchioloalveolar carcinoma Acinar adenocarcinoma Papillary adenocarcinoma

carcinoma Acinar adenocarcinoma Papillary adenocarcinoma internal control was positive. Discussion Fig. 2. Normal

internal control was positive.

Discussion

Fig. 2. Normal lung alveolar epithelial cells showing nuclear positivity for TIFt (arrows) . In the center, nests of cells from

a

metastatic

adenocarcinoma

without

nuclear

staining.

(Avidin-biotin-peroxidase/Diaminobenzidine x400)

adenocarcinomas were all solid carcinomas with mucus formation (Table 3). In all cases, the adjacent lung tis- sue presented immunoreactivity for TTFI in the nuclei of type II pneumocytes and Clara cells (Fig. 2).

Evaluation of the diagno5tic accuracy with and without

ITFl

When

the

pathologists correlated the TTF I im-

munostaining with the morphological data, the five cases diagnosed as primary lung adenocarcinomas were confirmed. Among the 8 cases in which differentiation

between metastatic and primary adenocarcinoma was

Primary lung adenocarcinomas are one of the most prevalent causes of metastatic disease with an unknown 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, 9, II/. The distinction hetween primary and secondary adenocarcinomas affecting the lungs is a difficult task, especially when we have a small biopsy or fine needle aspiration material. Recently, several reports have been published con- cerning new immunohistochemical antibodies against proteins and tissue-specific transcription factors as markers to distinguish primary from metastatic lung adenocarcinomas [l, 5, 6, 9, 10]. TTFI is considered one of the best markers to distinguish primary from metastatic primary lung adenocarcinomas II , 5, 6, 10}; besides, it is commercially available and able to be em- 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 (# of adenocarcinomas)

Extrapuhnonary tumors

Sensitivity (%)

Specificity (%)

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

Ordone z NG {9J 40 (40)

145

Borrin ski et al. [10/

18 (18)

12

67

100

Pre

se nt study

13 (13)

21

61.5

100

*: studies in which only primary lung tumors were evaluated

The sensitivity of TTFI in lung adenocarcinomas in

the different reported series ranged from 57.5 % to 76% (Table 4) [I, 2, 3, 4, 5, 6, 8,9, 1Oj. Excluding thyroid neoplasms, the TTFI specificity ranged from 99% {4/

to 100% for primary lung carcinomas [1 , 2, 3, 5, 6, 8,

9]. In our series, the sensitivity was 61.53% and the specificity 100%. Five cases of primary lung carcino- mas werc negative for TTFI; in all these cases, the au- thors observed that they were solid adenocarcinomas with mucin production. Among the TTF I primary lung adenocarcinomas, most of them were acinar adenocar- cinomas and only one solid adenocarcinoma with mucin production was positive for TTFI. These find- ings are in agreement with those published by Kauf- mann et a1. [II , who reported a TTFI sensitivity of 10% for mucinous lung adenocarcinomas. The authors also evaluated TTFl's role in the im- provement of the morphological diagnosis of primary lung adenocarcinomas. Without TTFI the ability of the pathologists to diagnose an adenocarcinoma accurately as a primary lung malignancy was just 38.46%. After TTFI, it improved to 61.53 %. Furthermore, one case misdiagnosed as a metastasis based solely on morpho- logical grounds could be correctly diagnosed as a pri- mary lung adenocarcinoma using TTFI. Another interesting feature of the present study is the ability of TTFI to infer a pulmonary origin for un- known primary site adenocarcinomas in which mor- phology does not allow the distinction between primary and metastatic lung adenocarcinomas. In the present se- ries, in 8 cases a distinction between primary and metastatic adenocarcinoma could not be achieved; with TTFL two of these cases could be diagnosed as pul- monary malignancies, lacking any clinical data. In conclusion, TTFI is a good marker to prove or to exclude the lung origin of adenocarcinomas with a sen- sitivity of61.53% and 100% specificity (excluding thy- roid neoplas m). However, is important to stress that

even primary lung adenocarcinomas presenting mucin production arc usually negative for TTFI. Taking this aspect into account, we suggest that TTFI be incorpo- rated in a panel for the differential diagnosis of adeno- carcinomas with an unknown primary site, mainly in small specimens and in fine needle aspiration biopsies.

References

I.

Kaufmann O. Dietel M (2000) Thyroid transcription fac- tor-I is the superior immunohistochemical marker for pulmonary adenocarcinomas and large cell carcinomas compared to surfactant proteins A and B. Histopathology

36:8-16

2.

Khoor A. Whitsett JA , Stahlman MT, Olson SJ, Cagle IT (1999) Utility of surfactant protein B precursor and thy- roid transcription factor I in differentiating adenocarcino- ma of the lung from malignant mesothelioma. Hum

Pathol 30: 695-700

3.

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4.

Bejarano PA. Baughman RP, Biddinger PW, Miller MA,

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oftj ss ue specific transcription factor-l

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58-02

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Received in revi sed version: August 3 ,2000 Accepted: Augu st 3, 2000