TTF-1 and Napsin A Do Not Differentiate Metastatic Lung Adenocarcinomas From Primary Esophageal Adenocarcinomas: Proposal of a Novel Staining Panel

Biopsy and resection specimens of 24 primary esophageal adenocarcinomas and 24 primary pulmonary adenocarcinomas were identified in our database. Institutional review board approval was received for the study. None of the patients diagnosed with primary pulmonary adenocarcinoma had a known history of primary esophageal adenocarcinoma and vice versa for patients diagnosed with primary esophageal adenocarcinoma. Tumors of the esophagus were selected based on confirmation from gross prosection or endoscopy of a distinctly esophageal location of the mass. Tumors of the gastroesophageal junction were excluded based on the premise that gastric adenocarcinomas extending into the esophagus could have a distinct immunohistochemical profile from that of pure esophageal adenocarcinomas. We purposefully selected mostly moderately differentiated pulmonary and esophageal adenocarcinoma cases because poorly differentiated carcinomas often lose immunohistochemical marker expression.

Formalin-fixed, paraffin-embedded 4-μm-thick tissue biopsy sections and resection specimens of 24 primary esophageal adenocarcinomas and 24 primary pulmonary adenocarcinomas were air dried and heated on a hot plate for 1 hour at 60°C. Epitope retrieval was achieved by heating the deparaffinized tissue slides for 20 minutes in Envision Flex Target Retrieval Tris-EDTA pH 9.0 (Dako, Carpinteria, California) using a steamer. Following 20 minutes of cooling, the tissue sections were separately incubated with the immunohistochemical stains TTF-1, N-cadherin, CDX2, IMP3, and napsin A for 20 minutes. For a list of primary antibodies, antigen retrieval, and secondary visualization methods, see Table 1. The detection system used was the Envision Flex Link HRP-DAB (Dako) following the manufacturer instructions. All staining steps were completed on the Dako Auto Stainer Plus.

Immunohistochemical staining was scored in a semiquantitative manner. The percentage of cells stained was graded from 0 to 4+ as 0, no staining; 1+, 1% to 25%; 2+, 26% to 50%; 3+, 51% to 75%; and 4+, 76% to 100%;

Intensity of staining by TTF-1 and CDX2 were graded using the following scale: 1+, weak; 2+, moderate; and 3+, strong (Table 2). The composite score was derived by the summation of the scores of the percentage of cells stained and the intensity of staining (Table 3). Staining for TTF-1 was restricted to nuclear positivity, with all other forms of staining, such as cytoplasmic, perinuclear, or Golgi pattern, considered negative.

More esophageal adenocarcinoma cells were positive when stained with CDX2 and IMP3 compared with lung adenocarcinoma. CDX2 was focally positive in esophageal tumors in 92% of cases (22 of 24), whereas 79% of lung primaries were negative. Staining with IMP3 had similar results, with 96% of esophageal tumors (23 of 24) being at least focally positive versus 63% of pulmonary tumors (15 of 24) being negative. Conversely, all esophageal adenocarcinomas (100%; 24 of 24) were negative with N-cadherin versus 42% of lung adenocarcinomas (10 of 24), which showed some staining. Pulmonary tumors exhibited staining patterns with TTF-1, napsin A, p63, and 34βE12, which were not significantly different from esophageal sites.

Esophageal tumor nuclei had more intense staining with CDX2 compared with lung tumors, with 88% (21 of 24) versus 21% (5 of 24) showing moderate or strong intensity, respectively. Similar findings were observed with IMP3 with 75% (18 of 24) versus 29% (7 of 24) having at least a moderate degree of staining intensity. N-cadherin was only positive in lung cases; therefore, any positivity was significant. Although the percentage of tumor cells positive for TTF-1, napsin A, p63, and 34βE12 were not significantly different based on tumor site, the pulmonary cases were more likely to have a moderate to strong degree of staining intensity compared with esophagus: 92% (n = 22) versus 54% (n = 13), 83% (n = 20) versus 50% (n = 12), 46% (n = 11) versus 17% (n = 4), and 92% (n = 22) versus 75% (n = 18), respectively.

Esophageal adenocarcinomas had higher composite scores than did pulmonary tumors with CDX2 and IMP3. Using N-cadherin, any composite score greater than 0 was indicative of a lung primary. Lung adenocarcinomas had slightly higher composite scores than did esophageal cases when using TTF-1, napsin A, p63, and 34βE12. Although the composite scores were somewhat increased in lung tumors, a significant proportion of esophageal tumors exhibited similar staining to lung adenocarcinomas using these 3 immunostains.

Statistically determined, composite-score cutoff points using univariate logistic regression analysis are shown in Table 4. Significant differences between esophageal and pulmonary tumor staining were identified for CDX2, IMP3, N-cadherin, napsin A, p63, and TTF-1.

Using the cutoff values in Table 4 in multivariable logistic-regression analysis, only 3 stains had significant differences in composite score between these 2 malignancies: CDX2, IMP3, and N-cadherin. Using this statistically determined immunohistochemical panel, esophagus adenocarcinomas were classified correctly in 23 out of 24 cases (96%), and lung adenocarcinomas were classified as pulmonary primary in 22 out of 24 cases (92%). Adding TTF-1, napsin A, and/or p63 to that panel did not change the classification of these tumors.

TTF-1 is a homeodomain protein that is expressed in the lung and trachea, and it appears to be integral in lung morphogenesis. For example, when TTF-1 translation is suppressed in the embryonic mouse, no pulmonary epithelium is produced.⁴  It is also expressed in rat and human tracheoesophageal fistulas.^5–7  Napsin A is a pepsinlike aspartic proteinase in the A1 clan of the AA clade of proteinases. Napsin A is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules, and pancreatic exocrine glands and ducts. Recent studies have demonstrated that napsin A is a sensitive and specific marker for pulmonary adenocarcinoma in primary and metastatic sites, especially when used in conjunction with TTF-1.⁸ 

Gastrointestinal pathology texts frequently state that esophageal adenocarcinomas should be negative with TTF-1.⁹  However, to the best of our knowledge, no prior studies have compared the immunohistochemical profiles of esophageal and pulmonary adenocarcinomas. Other studies, however, have shown a variety of carcinomas that demonstrate aberrant TTF-1 immunoreactivity, including breast, colon, and bladder.^10–12  We show that lung adenocarcinomas indeed do stain with a greater percentage of positive cells and greater staining intensity than do esophageal adenocarcinoma, although the difference is minimal. Greater than 50% of esophageal adenocarcinomas show moderate to strong staining intensity in more than half of tumor cells using TTF-1 and napsin A. Despite the minor statistical difference seen using TTF-1 and napsin A, their utility in differentiating lung and esophagus tumors is no better than flipping a coin. Therefore, TTF-1 and napsin A appear to have no significant value in this setting.

CDX2 has been reported to be positive in 10% to 20% of pulmonary and 80% of esophagus adenocarcinomas.^13–15  We demonstrated at least focal positivity in 92% of esophageal (22 of 24) and 21% of pulmonary tumors (5 of 24). CDX2 is a critical stain to include in any panel differentiating between lung and esophagus primary sites.

IMP3 is an oncofetal protein highly expressed in fetal tissue and malignant tumors but rarely found in adult, benign tissues. A previous study¹⁶  found IMP3 to be a highly sensitive and specific biomarker for the diagnosis of invasive esophageal adenocarcinoma and high-grade dysplasia, whereas it is negative in adjacent benign, squamous, and glandular mucosa.

In this study, IMP3 had a composite score of at least 4 in 96% of esophagus cases (23 of 24) versus 21% of lung specimens (5 of 24). IMP3 displayed the highest rate of positivity in primary esophageal adenocarcinomas compared with all other stains investigated. Thus, it discriminated between these 2 entities better than any of the other stains, even CDX2. IMP3 should be included in staining panels to differentiate between esophagus and lung primaries. Failure to positively stain with IMP3 is evidence against a primary esophageal adenocarcinoma.

N-cadherin is a member of the cadherin family of cell adhesion molecules.¹⁷  N-cadherin was negative in all cases (100%; 24 of 24) of primary esophageal adenocarcinoma. It showed at least focal positivity in 42% of primary pulmonary adenocarcinomas (10 of 24). In contrast to IMP3, N-cadherin positivity virtually excludes a diagnosis of primary esophageal adenocarcinoma. N-cadherin should be included as a negative immunohistochemical marker for tumors of esophageal origin. Conversely, positive staining for N-cadherin would raise the possibility of a metastatic pulmonary adenocarcinoma.

In most esophageal adenocarcinomas and half of pulmonary tumors, p63 was negative. In cases where staining was noted, the intensity was generally strong. Given inconsistency in positive staining and similar staining intensity, p63 staining is not a useful diagnostic adjunct. No significant staining patterns were identified with 34βE12 in either esophagus or lung primaries. Therefore, 34βE12 fails to discriminate between esophageal adenocarcinoma and pulmonary adenocarcinoma.

Our observations of the above stains were born out with biostatistical examination. Multivariate logistical-regression analysis showed that the optimal immunohistochemistry panel to distinguish esophagus from lung adenocarcinomas should consist of CDX2, IMP3, and N-cadherin. Despite the popular assertions that TTF-1 and napsin A are specific for lung primaries, the addition of these stains did not improve the diagnostic utility of this proposed panel.

In summary, TTF-1 positivity is significantly expressed in primary esophageal tumors in both the percentage of cells stained and the nuclear staining intensity, although not nearly to the extent and degree seen in primary pulmonary adenocarcinomas. These findings are contrary to presumptions that have been propagated in multiple literature sources broaching the subject of TTF-1 positivity in esophageal specimens. Instead of relying on TTF-1 and/or napsin A, an immunohistochemical panel consisting of CDX2, IMP3, and N-cadherin has shown excellent statistical and practical utility for reliable differentiation between these two tumor types.