Thyroid transcription factor 1

A marker for lung adenocarcinoma in body cavity fluids

Authors


Abstract

BACKGROUND

Adenocarcinomas are the most common epithelial malignancies in body cavity fluids. Subclassification of adenocarcinomas according to primary site can be a challenging task. Thyroid transcription factor 1 (TTF-1) is a nuclear transcription factor that is expressed in normal lung, in thyroid, and in their neoplasms. Because thyroid carcinomas rarely metastasize to the serosal surfaces, the authors used TTF-1 as a marker to distinguish adenocarcinomas of the lung from carcinomas of other organs.

METHODS

The authors studied 113 body cavity fluids (92 pleural fluid samples and 21 ascitic samples) from 113 patients with a diagnosis of adenocarcinoma on the basis of routine cytology. The primary sites of origin were confirmed clinically or histologically for all patients. There were 39 adenocarcinomas of the lung, 24 adenocarcinomas of the breast, 34 adenocarcinomas of the genitourinary tract, and 16 adenocarcinomas of the gastrointestinal tract. Archival Papanicolaou-stained, cytocentrifuged slides were used without destaining for immunocytochemistry with the monoclonal TTF-1 antibody using a commercial available method.

RESULTS

TTF-1 was expressed in 21 of 39 lung adenocarcinomas (54%). Intense nuclear staining was present in tumor cells that occurred in groups or in isolated form. In contrast, none of the other types of adenocarcinomas expressed TTF-1.

CONCLUSIONS

TTF-1 is a highly specific marker for adenocarcinomas of the lung in body cavity fluids. Immunocytochemistry using this antibody can be performed easily on archival Papanicolaou-stained, cytocentrifuged slides of fluid specimens. Cancer (Cancer Cytopathol) 2002;96:289–93. © 2002 American Cancer Society.

Adenocarcinomas are the most common epithelial malignancies found in body cavity fluids. Common primary sites include the lung, breast, gastrointestinal tract, and genitourinary tract. Identification of the specific site of origin may carry important prognostic as well as therapeutic implications. However, determination of the primary site of adenocarcinoma based on cytomorphology alone can be a challenging task. Tissue specific immunohistochemical markers offer an attractive means for confirming metastatic disease in patients with known primary tumors and may assist in identifying the primary sites in patients with adenocarcinomas of unknown origin.

Thyroid transcription factor 1 (TTF-1) is a homeodomain nuclear transcription protein that is expressed selectively in the epithelial cells of the thyroid and lung.1–4 It mediates transcription of thyroid specific genes in follicular cells and activates the transcription of surfactant protein genes in pulmonary alveolar cells.1, 4 Immunohistochemistry of human tumors has demonstrated TTF-1 staining in tumors derived from lung or thyroid.5–7 Some investigators have used a monoclonal TTF-1 antibody on thyroid neoplasms and found an overall immunoreactivity of 74%.8 Others have used TTF-1 as a tissue specific marker to distinguish adenocarcinomas of the lung from other malignancies that may have overlapping morphologic features.7, 9–11 Using paraffin embedded material, a sensitivity of up to 76% and a specificity of 100% have been reported for adenocarcinomas of lung origin compared with mesotheliomas and adenocarcinomas of breast, ovary, colon, prostate, and renal cell origin. We used archival Papanicolaou-stained, cytocentrifuged slides of body cavity fluids and investigated the value of a commercially available monoclonal TTF-1 antibody to distinguish adenocarcinomas of the lung from those of other organs.

MATERIALS AND METHODS

Samples

A computer-generated list of all body cavity fluid specimens from patients who were diagnosed with adenocarcinoma by the Department of Cytopathology at the University of Miami/Jackson Memorial Medical Center between 1995 and 2000 was obtained. The selection of patients was restricted to include those in whom the primary site of origin for the carcinoma was confirmed based on clinical findings (n = 39 patients) or histopathologic findings (n = 74 patients) (Table 1). None was of thyroid origin. The 113 body cavity fluids collected from 113 patients included 92 pleural fluids and 21 ascitic fluids. Four glass slides that were stained previously by a routine Papanicolaou method were retrieved for each patient. The diagnosis of adenocarcinoma based on cytomorphology was confirmed. Classic cytologic features of malignancy included three-dimensional aggregates comprised of cells with increased nuclear-to-cytoplasmic ratios, irregular nuclear membranes, coarse chromatin, and large irregular nucleoli. Single, isolated, morphologically abnormal cells were large and pleomorphic compared with the background mesothelial cells. Patients who had a diagnosis of adenocarcinoma that was equivocal based on a routine Papanicolaou stain were excluded from the study. The sensitivity and specificity of TTF-1 in fluid cytology for primary pulmonary lung adenocarcinomas, compared with other adenocarcinomas, were calculated. The positive and negative predictive values of TTF-1 for adenocarcinomas of lung origin using archival Papanicolaou-stained slides also were calculated.

Table 1. Primary Site of Adenocarcinomas in 113 Body Cavity Fluids
Site of originHistologic diagnosis (n = 74 patients)Clinical history (n = 39 patients)
Pleural fluidAscitic fluidPleural fluidAscitic fluid
Lung130260
Breast18060
Stomach2010
Colon3020
Appendix0100
Pancreas3012
Gallbladder0100
Uterus3200
Ovary111310
Fallopian tube1000
Peritoneal carcinoma0200
Prostate1000

Antibody

The anti-TTF-1 antibody (clone 8G7G3/1; Dako Corp. Carpinteria, CA) is a mouse monoclonal immunoglobulin G1 antibody. The immunostaining pattern with anti-TTF-1 is exclusively nuclear.

Immunoperoxidase Procedure

Slides from previously Papanicolaou-stained, cytocentrifuged preparations of body cavity fluids were used for immunostaining. Destaining was not necessary. The coverslips were removed, and the slides were hydrated in decreasing ethanol solutions. Endogenous peroxidase was blocked with 6% hydrogen peroxidase for 3 minutes. The slides were rinsed in water, placed in a dish containing a target retrieval solution (Dako) that was heated previously to 90 °C, then placed in a steamer for 20 minutes to achieve optimal antigen retrieval. After a 20-minute cooling period, the slides were incubated with the avidin solution and, subsequently, with the biotin solution using the Biotin Blocking System (Dako) prior to application of the primary antibody. The TTF-1 monoclonal antibody (clone 8G7G3/1; Dako) was used at a dilution of 1:150 and incubated with the samples for 30 minutes. The slides were then rinsed in buffer and incubated for 25 minutes with the linking solution (LSAB+ kit; Dako; biotinylated antimouse, antirabbit, and antigoat). This was followed by a rinse in buffer and incubation with streptavidin peroxidase for 25 minutes. After rinsing in buffer, the slides were submerged in 3,3′-diaminobenzidine tetrahydrochloride for 5 minutes. One percent cupric sulfate was applied for 2 minutes, and the slides were counterstained with 0.1% Fast Green. The slides were then dehydrated through gradient alcohols, cleared in xylene, and coverslipped. A section of normal thyroid was used as the positive antibody control. The results of TTF-1 immunostaining were based on the nuclear staining of tumor cells. Staining was considered positive if > 10% of tumor cell nuclei reacted with any intensity.

RESULTS

The results of TTF-1 immunostaining of adenocarcinomas in body cavity fluids are shown in Table 2. TTF-1 was expressed in 54% of primary lung adenocarcinomas. Positive immunoreactivity for TTF-1 was characterized by a dark-brown, diffusely granular nuclear staining in tumor cells that occurred singly or in groups (Figs. 1, 2). Intense nuclear staining for TTF-1 was evident clearly on cytocentrifuged slides containing only a few tumor cells (Fig. 2). Variations in nuclear staining intensity occurred in the thicker smears of highly cellular samples.

Table 2. Thyroid Transcription Factor 1 Immunostaining of Adenocarcinomas in Cytocentrifuge Preparations of Body Cavity Fluids
Site of originTotal (n = 113 patients)No. TTF-1 positive (%)
  1. TTF-1. thyroid transcription factor 1.

Lung3921 (54)
Breast240 (0)
Gastrointestinal/biliary tract160 (0)
Genitourinary tract340 (0)
Figure 1.

(A) Cellular evidence of adenocarcinoma in pleural fluid from a female patient age 64 years with a lung mass and a history of estrogen receptor (ER) positive breast carcinoma (cytospin preparation; Papanicolaou stain). (B) Positive immunostaining for thyroid transcription factor 1 (TTF-1) and negative staining for ER in tumor cells identifies lung as the primary site (cytospin, TTF-1; Dako, Carpinteria, CA).

Figure 2.

(A) Pleural fluid from a male patient age 57 years with lung adenocarcinoma and malignant effusions. Only a few malignant cells were present (cytospin; Papanicolaou stain). (B) Sharp, intense nuclear staining highlights the tumor cells despite their limited number (cytospin preparation; thyroid transcription factor 1; Dako).

The specificity and positive predictive values of the TTF-1 antibody for the adenocarcinomas of lung origin in our series were both 100%. None of the other types of adenocarcinomas in the body cavity fluids expressed TTF-1. Reactive mesothelial and inflammatory cells that were present on the cytocentrifuged smears, likewise, were negative for TTF-1. The negative predictive value of TTF-1 for primary pulmonary adenocarcinomas using Papanicolaou-stained, cytocentrifuged smear preparations was 80%. Eighteen of 39 fluids from adenocarcinoma of lung origin were negative for TTF-1. Rare artifacts included nonspecific cytoplasmic membrane staining seen predominantly in cell balls or tightly cohesive tridimensional groups (Fig. 3).

Figure 3.

(A and B) Nonspecific cytoplasmic staining may be seen occasionally in tridimensional clusters of malignant cells (cell balls). This represents an artifact and can be a potential pitfall (cytospin preparation; thyroid transcription factor 1; Dako).

DISCUSSION

Adenocarcinoma is the most common cause of malignancy in body cavity fluids. Usual primary sites include the lung, breast, gastrointestinal tract, and genitourinary tracts. Predicting the site of origin of an adenocarcinoma can be difficult due to overlapping morphologic characteristics.

Organ specific markers using immunohistochemistry offer an attractive means for subclassifying adenocarcinomas according to primary site. TTF-1 is a tissue specific protein that is expressed selectively in the epithelial cells of the thyroid and lung.1–4 Studies using antibodies generated against TTF-1 have demonstrated immunostaining of carcinomas derived from thyroid and lung but not from breast, ovary, kidney, colon, or prostate.5–7 Using formalin fixed, paraffin embedded histologic material, it has been shown that TTF-1 is a sensitive marker for primary tumors of the thyroid, with an overall immunoreactivity of 74%.8 Similarly, antibodies to TTF-1 have demonstrated a sensitivity up to 76% and a specificity of 100% for lung adenocarcinomas compared with mesotheliomas and adenocarcinomas of the breast, colon, and prostate on formalin fixed, paraffin embedded tissue.7, 11

Lung is a common primary site for adenocarcinomas in body cavity fluids.12–14 We investigated the use of TTF-1 to distinguish adenocarcinomas of lung origin from other relatively common adenocarcinomas in 113 body cavity fluid samples. Using a commercially available monoclonal antibody for TTF-1, we demonstrated a sensitivity of 54% and a specificity of 100% for lung adenocarcinomas compared with adenocarcinomas from breast, the gastrointestinal tract, and the genitourinary tract. Included among the patients with nonpulmonary adenocarcinomas were three patients with gastric adenocarcinoma, six patients with pancreatic adenocarcinoma, eight patients with adenocarcinoma of the female genital tract (including two patients with adenocarcinoma of endometrial origin), one patient with appendiceal adenocarcinoma, and one patient with adenocarcinoma of the gallbladder. All samples from these patients were clearly negative for TTF-1. Although TTF-1 has been reported as notably negative in several series of patients with adenocarcinoma of the breast, ovary, colon, kidney, and prostate, less has been written about the reactivity of TTF-1 in adenocarcinomas arising from less common sites. In a study that focused primarily on the value of TTF-1 to distinguish primary lung adenocarcinomas from metastatic breast carcinoma, Bejarano et al. reported focal, positive immunoreactivity for TTF-1 in 1 of 66 gastric adenocarcinomas and in 1 of 8 endometrial adenocarcinomas.11 TTF-1 is also a marker of thyroid neoplasms,8 although these rarely metastasize to the serosal surfaces.13, 14 For those patients with a positive effusion who have a history or clinical suspicion of a thyroid primary tumor, the additional use of thyroglobulin as a thyroid specific immunohistochemical marker may be helpful. There were no patients with adenocarcinoma of thyroid origin in our study.

Two previous studies used cell block preparations of cytologic specimens to evaluate the expression of TTF-1 in primary lung carcinomas. Di Loreto et al. reviewed the positive bronchial and sputum cytology specimens from 58 patients with lung carcinoma.15 Immunostaining for TTF-1 was detected in 5 of 8 patients (62.5%) who had a diagnosis of adenocarcinoma. Harlamert et al. used cell block preparations of lung aspirates, pleural fluids, and bronchial washes and lavages from patients with primary lung and metastatic breast carcinomas to evaluate the expression of several immunocytochemical markers, including TTF-1.10 Their study consisted of 41 primary lung carcinomas of different subtypes and 6 metastatic breast adenocarcinomas. TTF-1 was detected in 16 of 21 primary lung adenocarcinomas (76%). None of the breast adenocarcinomas stained positive for TTF-1.

The current study was designed to determine immunoreactivity for TTF-1 using archival Papanicolaou-stained, cytocentrifuged slides. One of the advantages of using this material is that immunocytochemistry studies can be performed on the same cells that are used for diagnosis and provide an important ancillary tool, even when the diagnostic cells in the fluid are few. In this study, the slightly lower sensitivity of TTF-1 for lung adenocarcinomas compared with the sensitivity reported previously using paraffin embedded material may have been the result of decreased exposure of the intranuclear TTF-1 antigen in the cytocentrifuged preparations. Conversely, the high specificity of TTF-1 for adenocarcinomas of known lung origin in our study was similar to the specificity reported by others and supports the use of TTF-1 as a valuable immunocytochemical marker in body cavity fluid cytology.

The immunostaining pattern with the TTF-1 antibody consists of a diffuse, finely granular, dark-brown nuclear stain visualized in tumor cells. The nuclear immunostaining generally is intense and contrasts with the negative reaction of the nuclei of macrophages, lymphocytes, neutrophils, and mesothelial cells seen in the background. The distinct, sharp pattern of nuclear reactivity allows for clear evaluation of immunostaining results, even in specimens with limited diagnostic cells.

Nonspecific cytoplasmic staining of tumor cells by TTF-1 in tridimensional groups and cell balls was encountered as a rare artifact. Entrapment of immunologic reagents with artifactual staining of large cell groups has been described as a potential cause of false positive results in fluid cytology.16, 17 The evaluation of positivity in such specimens should be limited to single cells or two-dimensional groups with diffuse nuclear staining.

The application of the TTF-1 immunocytochemical marker onto previously Papanicolaou-stained slides of adenocarcinoma can be very helpful in daily clinical cytology practice. The immunocytochemical technique is simple, and the TTF-1 monoclonal antibody yields highly specific and relatively sensitive results. A positive reaction for TTF-1 may serve to confirm the spread of a known primary lung tumor, establish the recurrence of a previously treated adenocarcinoma of the lung, or identify the lung as a possible source of malignancy in a patient with an unknown primary tumor site. The use of cytocentrifuged preparations together with the distinct, intense nuclear immunostaining pattern of TTF-1 allows for clear and adequate results even in specimens with a limited number of diagnostic cells. Finally, TTF-1 is a marker of thyroid as well as lung adenocarcinoma. Therefore, caution should be exercised in patients with a known history of thyroid carcinoma who present with a lung mass and a malignant effusion. In such patients, the immunostaining results for TTF-1 should be evaluated in combination with other thyroid markers, such as thyroglobulin, to exclude the rare possibility of thyroid carcinoma metastatic to the serosal surface.

Acknowledgements

The authors are grateful to Ms. Estela Garcia-McDougal, M.P.A., S.C.T. (A.S.C.P.), for her assistance with this article.

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