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Keywords:

  • cytology;
  • Diff-Quik method;
  • fine-needle aspiration;
  • immunoperoxidase technique;
  • lung neoplasms;
  • Papanicolaou technique;
  • thyroid transcription factor-1

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND

Fine-needle aspiration (FNA) is used commonly for the diagnosis of pulmonary neoplasms. It has been reported that thyroid transcription factor-1 (TTF-1) is a sensitive and specific marker for certain primary lung tumors. To the authors' knowledge, the use of TTF-1 immunostaining on FNA smears has not been documented in the literature. This study was designed to examine the utility of TTF-1 immunostaining on FNA specimens from various types of lung tumors by comparing the expression rates on Papanicolaou (Pap)-stained and Diff-Quik (DQ)-stained smears with the expression rates on cell block (CB) sections.

METHODS

Forty-three FNA specimens of lung tumors were studied, including 34 primary tumors (14 adenocarcinomas, 12 squamous carcinomas, and 8 small cell carcinomas) and 9 metastatic tumors. One Pap-stained slide and one DQ-stained slide were selected from each tumor. The cytologic material from the slides was transferred to positively charged slides. Unstained recuts were obtained from the CB sections. All slides were stained with TTF-1 monoclonal antibody using heat-induced epitope retrieval and a labeled polymer detection system.

RESULTS

Twelve of 14 pulmonary adenocarcinomas were positive for TTF-1 (11 specimens on both CB sections and Pap-stained smears and 1 specimen on all 3 preparations, including the DQ-stained smear). Two of 14 adenocarcinomas were negative for TTF-1. Of 12 pulmonary squamous carcinomas, only 1 was positive for TTF-1 (on the CB section and the Pap-stained smear); the others were negative in all 3 preparations. Of eight small cell lung carcinomas, six specimens showed positive staining for TTF-1 on both CB and Pap-stained preparations; one of those also was positive on the DQ-stained smear. The remaining two small cell lung carcinomas were negative for TTF-1 in all three preparations. All metastatic tumors were negative for TTF-1.

CONCLUSIONS

TTF-1 immunostaining of pulmonary neoplasms was applicable to FNA smears that were stained previously with the Pap technique, and the rate of positive staining in each tumor type was identical to the rate of positive staining in CB sections and was comparable to that reported in previous publications. Smears previously stained with the DQ method were unreliable for TTF-1 immunostaining. Cancer (Cancer Cytopathol) 2004;102:000–000. © 2004 American Cancer Society.

Thyroid transcription factor-1 (TTF-1) is a nuclear protein that is expressed almost exclusively in the normal thyroid and lung and in most carcinomas derived from those organs. TTF-1 is a 38-kilodalton (kD) member of the NKx2 family of homeodomain transcription factors. In the normal lung, TTF-1 regulates the transcription of lung-specific genes for surfactant and Clara cell secretory proteins.1 The value of TTF-1 immunostaining has been investigated in histologic sections of surgical specimens and cell block (CB) sections of cytologic material. The results have demonstrated that TTF-1 is both a sensitive and specific marker for certain types of primary lung tumors.2–7

Fine-needle aspiration (FNA) is used frequently in the diagnosis of lung lesions. It is preferred especially when surgery may not be the primary treatment option (e.g., in the diagnosis of small cell lung carcinoma [SCLC] and metastatic lung tumors). Although 95% ethanol-fixed, Papanicolaou (Pap)-stained smears and air-dried, Diff-Quik (DQ)-stained smears are used for light microscopic diagnosis in routine cytology practice, pathologists prefer using CB preparation for immunostains because the procedure is similar to that of routinely fixed and processed histology sections. However, the amount of material obtained during FNA of the lung frequently is limited because of the risk of pneumothorax. There is often insufficient material for CB preparation.

To our knowledge, the reliability of TTF-1 immunostaining using routinely fixed and stained FNA smears from lung tumors has not been investigated fully. The current study was designed to compare TTF-1 immunostaining on Pap-stained and DQ-stained FNA smears with TTF-1 immunostaining on CB sections and to evaluate the diagnostic application of TTF-1 in various types of lung tumors.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Study Material

We reviewed our computer files for a 5-year period and found 43 FNA specimens (all from different patients) of lung neoplasms with both Pap-stained and DQ-stained smears and CB sections available. Of the 43 specimens, 34 specimens were primary pulmonary carcinomas and 9 were metastatic tumors. Of 34 primary lung tumors, 14 were adenocarcinomas, 12 were squamous carcinomas, and 8 were SCLC. The nine metastatic tumors included adenocarcinomas from breast (three tumors), colon (two tumors), endocervix (one tumor), and stomach (one tumor) as well as one adenoid cystic carcinoma of the tongue and one melanoma. All FNA procedures were performed with computed tomography guidance by radiologists. Smears either were fixed in modified Carnoy solution (7 parts 95% ethanol in 0.5 parts glacial acetic acid) and stained with the Pap technique or were air dried and stained with the DQ method. Material for CB preparations was fixed in 10% neutral buffered formalin, subjected to routine histology processing, and then embedded in paraffin blocks. For this study, we selected one Pap-stained smear and one DQ-stained smear from each specimen. Unstained sections were obtained from the CB sections.

Immunochemistry

Cytologic material from the original smears was transferred to positively charged slides using the following procedure. The area containing diagnostic material was outlined on the coverslip with a marking pen and the corresponding lines were drawn on the underside of the slide with a diamond pen. The slides were soaked in xylene for coverslip removal, with additional immersion in xylene to ensure complete removal of mounting media after removing the coverslips. The slides then were completely covered with liquid coverglass medium (Mount Quick; Newcomer Supply, Middleton, WI) and placed horizontally in a 60 °C oven for 2 hours to allow the coverglass medium to dry and harden. The resulting slides were immersed in a 45 °C water bath for at least 1 hour or as long as necessary to easily pry off the medium from the edges of the slides using a scalpel blade. The sliver on each slide then was divided into segments corresponding to the scoring of the underside of the slide prior to final removal. After moistening with water, each segment was placed on a separately moistened, positively charged slide and was mounted on the same side as the original slide. The slides then were dried in a horizontal position in a 60 °C oven for 1 hour.

Prior to immunostaining, the coverglass medium was removed by immersing the slides in 3 changes of xylene for 4 minutes each time. The slides then were hydrated by sequential immersion in absolute ethanol (twice), 95% ethanol, 70% ethanol, and water. Unstained sections from the CB sections were subjected to the same steps for removal of paraffin and rehydration.

The immunohistochemical procedures for the smears and the CB sections were identical. The process was initiated with application of heat-induced epitope retrieval using a vegetable steamer (Handy Steamer Plus; Black & Decker, Shelton, CT). The slides were placed in 1 mM ethylenediamine tetraacetic acid buffer (pH 8), steamed for 40 minutes, and then allowed to cool to room temperature in the buffer for 20 minutes. The Dako Autostainer (Dako Corporation, Carpinteria, CA) was used for the remaining steps. The slides were incubated in 0.9% hydrogen peroxide in methanol for 20 minutes to quench any endogenous peroxidase activity and were then incubated with a 1:50 dilution of TTF-1 antibody (clone 8G7G3/1; Dako Corporation) for 32 minutes at room temperature. The EnVision+ (Dako Corporation) detection system was used with diaminobenzidine as the chromogen. Tumors were considered positive for TTF-1 if > 10% of the tumor cells demonstrated nuclear staining.

Controls

Identically prepared slides from nonneoplastic lung and thyroid FNA smears and histologic sections from pulmonary adenocarcinoma and nonneoplastic lung tissue were used as positive controls. Negative controls consisted of FNA smears and CB sections in which Tris buffer was substituted for the primary antibody.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The mean patient age was 68 years (range, 44–77 years). Of 43 patients, 19 patients were women, and 24 patients were men. The diagnoses of primary lung carcinoma and metastatic tumor were made based on clinical, radiologic, and pathologic findings.

Controls

The alcohol fixed, Pap-stained FNA smears of nonneoplastic lung and thyroid and the histologic sections from nonneoplastic lung and pulmonary adenocarcinoma showed moderate to strong nuclear immunoreactivity for TTF-1. Air-dried, DQ-stained smears from lung specimens were nonreactive, and similarly stained smears from thyroid specimens showed weak nuclear immunoreactivity. Figure 1 illustrates the three types of control slides.

thumbnail image

Figure 1. Controls for thyroid transcription factor-1 immunostaining on (A) a thyroid fine-needle aspiration (FNA) smear previously stained using the Papanicolaou (Pap) method showing positive staining; (B) a thyroid FNA smear previously Diff-Quik (DQ) stained showing weakly positive staining; (C) a lung FNA smear previously Pap stained showing positive staining; (D) a lung FNA smear previously DQ stained showing no immunoreactivity; and (E) a formalin fixed, paraffin embedded lung adenocarcinoma tissue section showing positive staining. Original magnification × 400.

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Immunoreactivity for TTF-1 in Cytologic Material

The results of TTF-1 immunostaining in 34 specimens of primary lung carcinomas are shown in Tables 1–3 and Figure 2. TTF-1 positivity was observed in 12 of 14 adenocarcinomas, 6 of 8 SCLCs, and 1 of 12 squamous carcinomas and was detectable on both CB sections and Pap-stained smears in all the positive specimens. Material from air-dried, DQ-stained preparations was largely nonreactive for TTF-1, with the exception of one lung adenocarcinoma and one SCLC. All tumors that were positive for TTF-1 showed moderate to strong nuclear immunoreactivity. The poorly preserved areas of necrotic/degenerating SCLC cells or areas with crush artifact were negative or only weakly positive for TTF-1 (Fig. 3). TTF-1 immunoreactivity was not detected in any of the preparations from all nine metastatic lung neoplasms. No cytoplasmic staining was observed in any of the specimens.

Table 1. Thyroid Transcription Factor-1 Expression in Different Preparations of Fine-Needle Aspiration Specimens of Primary Lung Adenocarcinoma
CaseTTF-1 expression
CB sectionPap-stained smearDQ-stained smear
  1. TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; +: positive; −: negative.

1++
2
3++
4++
5++
6++
7++
8++
9
10++
11++
12++
13++
14+++
Table 2. Thyroid Transcription Factor-1 Expression in Different Preparations of Fine-Needle Aspiration Specimens of Small Cell Lung Carcinoma
CaseTTF-1 expression
CB sectionPap-stained smearDQ-stained smear
  1. TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; −: negative; +: positive.

1
2++
3++
4++
5
6+++
7++
8++
Table 3. Thyroid Transcription Factor-1 Expression in Different Preparations of Fine-Needle Aspiration Specimens of Primary Lung Squamous Carcinoma
CaseTTF-1 expression
CB sectionPap-stained smearDQ-stained smear
  1. TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; −: negative; +: positive.

1
2++
3
4
5
6
7
8
9
10
11
12
thumbnail image

Figure 2. Thyroid transcription factor-1 immunostaining in fine-needle aspiration (FNA) material from primary lung adenocarcinoma showing (A) positive staining on a previously Papanicolaou-stained FNA smear, (B) no immunoreactivity on a previously Diff-Quik-stained FNA smear, and (C) positive. staining on a cell block section. Original magnification × 400.

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thumbnail image

Figure 3. Crushed small cell lung carcinoma cells are negative or weakly positive for thyroid transcription factor-1 (TTF-1), whereas the intact tumor cells are positive for TTF-1 on a cell block section. Original magnification × 400.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A variety of neoplasms arise from the lung, and the lung also frequently is the site for metastatic neoplasms. Primary and metastatic lung tumors may share similar microscopic features. TTF-1 has recently been recognized for its utility in the diagnosis of certain types of primary lung carcinomas, most notably adenocarcinoma and SCLC. Based on the literature, the TTF-1 expression rate in histologic specimens ranges from 81–100% (mean, 89%) in SCLC and from 75–100% (mean, 82%) in adenocarcinoma.2–12 TTF-1 immunoreactivity on CB sections appears less, with a reported range of 33–93% (mean, 62%) in SCLC and 19–89% (mean, 62%) in adenocarcinoma.13–16

Although FNA is used frequently to obtain lesional material for the diagnosis of lung nodules, it is not always possible to obtain sufficient cytologic material for preparation of CB sections or to anticipate which FNA smear(s) will contain adequate cellularity prior to staining. Thus, immunostaining, if necessary, may have to be performed on cytologic smears that are fixed previously and stained with conventional cytologic methods, such as alcohol fixed, Pap-stained smears and air-dried, DQ-stained smears.

In the current study, nuclear immunoreactivity for TTF-1 was observed in 12 of 14 specimens (86%) of primary lung adenocarcinoma, 6 of 8 specimens (75%) of SCLC, 1 of 12 specimens (8%) of primary lung squamous carcinoma, and 0 of 9 specimens (0%) of metastatic neoplasms to the lung. None of the metastatic tumors were of thyroid origin. These results are similar to those reported in the literature. The TTF-1 positivity on CB sections always was accompanied by positivity on the smears that were stained previously with the Pap technique. In contrast, most smears that previously had been air-dried and stained with DQ did not show TTF-1 immunoreactivity.

TTF-1 immunoreactivity in primary lung adenocarcinoma is both sensitive and specific, with multiple investigations consistently showing that, except for thyroid carcinomas, extrapulmonary adenocarcinomas are nonimmunoreactive for this marker.2, 8–10, 13, 14 Distinguishing primary lung adenocarcinoma from metastatic adenocarcinoma is a frequent challenge because of the clinical, radiographic, and pathologic overlap in presentation. For therapeutic purposes, surgical resection usually is the treatment of choice for primary lung adenocarcinoma when the tumor is resectable; however, chemotherapy and/or other noninvasive procedure(s) may be indicated for metastatic disease. TTF-1 and other markers (e.g., cytokeratin 7, cytokeratin 20, thyroglobulin, and estrogen and progesterone receptors) now greatly facilitate this distinction.

SCLC reportedly has a consistently high expression rate of TTF-1 immunostaining on histologic sections. In contrast, 4 studies of TTF-1 immunostaining on CB sections resulted in a wide range of immunoreactivity (33–93%).13–16 In the current study, TTF-1 was detected in 6 of 8 specimens (75%) of SCLC, which is within the reported range. Both the quantity and the quality of FNA samples may contribute to the slightly lower expression rate of TTF-1 in cytologic specimens compared with surgical specimens.13 In general, the amount of cytologic material present, even in CB preparations, is usually less compared with the amount of material from surgical specimens. The current study also showed that poorly preserved tumor cells, due to necrosis or the crush artifact commonly seen in SCLC, more frequently were negative for TTF-1 immunostaining. In addition, patchy or focal expression of antigen by tumor cells as well as the inability to select the most optimal tissue section for immunocytochemistry, which is possible in surgical specimens, may contribute to a lower sensitivity for TTF-1 detection in cytologic specimens.11 It is important to note that the TTF-1 immunoreactivity has been observed in some of extrapulmonary small cell carcinomas, with a range of 11–80%.6, 11, 12, 17 Thus, unlike adenocarcinomas, TTF-1 cannot be used to distinguish SCLC from extrapulmonary small cell carcinoma. Nevertheless, TTF-1 may be helpful when it is used in conjunction with clinical and radiologic findings.

In the current study, primary lung squamous carcinomas had a TTF-1 expression rate of 8%. This rate is consistent with that found in previous studies using histologic or CB sections (mean, 9%; range, 0–38%).4, 9, 10, 13–16

TTF-1 is a nuclear marker. It is noteworthy that cytoplasmic staining with TTF-1 has been reported in 6.3% of carcinomas from various sites.18 In one study, 71% of hepatocellular carcinomas showed TTF-1 cytoplasmic staining.19 In addition, normal liver cells frequently show this cytoplasmic staining. The basis for this pattern of staining is not known. None of the specimens in the current study showed cytoplasmic staining.

Although the immunostaining technique can be applied directly to Pap-stained FNA smears, it is advantageous to transfer the cytologic material to positively charged slides to minimize cell loss if the original slides were not coated with adhesive. Furthermore, the material from a slide that contains high cellularity can be divided selectively into several portions and transferred to multiple slides so that more immunochemical markers may be investigated if needed. A destaining step is not necessary.

Utilizing air-dried, DQ-stained smears resulted in false negativity for TTF-1 in most of our specimens of lung adenocarcinoma and SCLC, and only weak immunoreactivity was noted in control specimens of thyroid cytologic material. A previous study demonstrated that air drying decreased immunoreactivity for AE1/AE3 and that the type of fixative affected immunoreactivity to some degree.20 Air-dried cells may exhibit an overall lower antigen density. It is unclear whether methanol, which is the fixative used for air-dried smears, affects TTF-1 immunoreactivity. Thyroid tissue may have a higher TTF-1 antigen density compared with lung tissue. Further studies are needed to assess whether this false-negative or weak immunoreactivity for TTF-1 on air-dried, DQ-stained smears can be corrected by extending the antibody and/or chromogen incubation time, by using a different epitope-retrieval technique, or by using reagents that are more sensitive.

In the current study, the immunoreactivity observed in the FNA material that was stained previously with the Pap technique was identical to the immunoreactivity observed in the CB preparations. The detection rates of TTF-1 in various lung neoplasms were within the ranges reported previously in the literature. FNA smears that have been air-dried and DQ stained should not be used for TTF-1 immunostaining. In addition, in certain circumstances, the smear transfer is necessary for facilitating the immunostaining on previously routinely fixed and stained cytologic smears.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Bonnie Price Whitaker, H.T. (ASCP), I.H.C., for her expert technical assistance in immunocytochemistry and immunohistochemistry.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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