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

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1 TTF-1 and Napsin A Do Not Differentiate Metastatic Lung Adenocarcinomas From Primary Esophageal Adenocarcinomas Proposal of a Novel Staining Panel Kanwaijit S. Aulakh, MD; Cary D. Chisholm, MD; Daniel A Smith, DO, PhD; V.O. Speights, DO Context. When adenocarcinomas arise within the esophagus, particularly when located away from the gastroesophageal junction, it may be important in some patients to differentiate between a primary esophageal adenocarcinoma and metastasis from another site. Lung adenocarcinoma is one tumor that has been reported to frequently metastasize to the esophagus. Objectives. To create a panel of immunohistochemical markers that can reliably distinguish between an esophageal and pulmonary primary; within the gastrointestinal pathology literature, including published articles and textbooks, common lung immunohistochemical markers, such as TTF-1, are assumed to be negative in esophageal adenocarcinoma, yet, to our knowledge, no study has yet investigated the veracity of that presumption. Design. In this study, 24 cases each of pulmonary and esophageal adenocarcinomas were stained with TTF-1, napsin A, CDX2, 34bE12, N-cadherin, and IMP3 in an attempt to define an optimal panel for differentiation. Esophageal adenocarcinomas occurring at the gastroesophageal junction were excluded in this study because a gastric primary tumor cannot be excluded in those cases. Results. Surprisingly, TTF-1 and napsin A were positive in similar proportions of tumors from both sites. Those markers that differentiated statistically between esophageal and pulmonary adenocarcinoma were IMP3, CDX2, and N-cadherin. Conclusions. When differentiating the origin of a tumor as either esophageal or pulmonary, an immunohistochemical panel consisting of IMP3, CDX2, and N- cadherin is superior to either TTF-1 or napsin A. (Arch Pathol Lab Med. 2013;137: ; doi: /arpa OA) Malignant tumors frequently involve multiple organ systems, either by direct extension or by metastasis. Moreover, adenocarcinomas from various sites can be morphologically similar, necessitating immunohistochemical examination to accurately determine the site of origin. Distinguishing a primary site when adenocarcinomas involve both the esophagus and lungs can be difficult. Tumors in these organs may metastasize or can invade via direct extension because of the close proximity of the involved organs. Accepted for publication October 3, From the Department of Pathology, Scott & White Healthcare, Temple, Texas. Dr Aulakh is now with the Department of Pathology, St John Medical Center, Tulsa, Oklahoma; Dr Smith is now with the Department of Pathology, Central Texas Pathology Laboratory, Waco; and Dr Chisholm is now with the Dermatopathology Laboratory, Cockerell & Associates, University of Texas, Southwestern Medical Center, Dallas. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Cary D. Chisholm, MD, Dermatopathology Laboratory, Cockerell & Associates, University of Texas, Southwestern Medical Center, 2330 Butler St, Ste 115, Dallas, TX ( cary. chisholm@gmail.com). Metastasis to the esophagus is not an uncommon occurrence. However, certain tumors have been documented to metastasize to the esophagus more frequently than others, including lung, thyroid, breast, and ovary. In a study of 1835 autopsy cases of patients who died from carcinomas, metastases to the esophagus occurred in 112 cases (6.1%). The lung was the most common primary site, representing 51 of 112 cases (45.5%) of esophageal metastases. Of these 51 cases, 32 (63%) were adenocarcinoma and 19 (37%) were squamous cell carcinoma. 1 Because of the histomorphologic similarities, distinguishing the exact site of a primary tumor can be exceedingly difficult and is critical because the treatment regimens differ depending on the tissue origin. Thyroid transcription factor-1 (TTF-1) protein has historically been a reliable marker of pulmonary adenocarcinoma and thyroid malignancies. Initially, the 8G7g3/1 clone of TTF-1 was reported to be positive in 76% of pulmonary adenocarcinomas. 2 However, recent studies comparing TTF- 1 with napsin A, have shown TTF-1 to stain positively in more than 60% of primary pulmonary adenocarcinomas. 3 Despite its ubiquitous use in the evaluation of primary pulmonary tumors and distant metastases, its expression in primary esophageal adenocarcinoma has never, to our knowledge, been thoroughly investigated. Neural cadherin 1094 Arch Pathol Lab Med Vol 137, August 2013 TTF-1 and Napsin A Utility in Esophageal Tumors Aulakh et al

2 Table 1. Immunohistochemical Stains Used Antibody Source Clone Dilution Antigen Retrieval Detection System TTF-1 Dako, Carpinteria, 8G7g3/1 Ready-to-use 34bE12 HMK Dako, Carpinteria, 34bE12 Ready-to-use N-cadherin Dako, Carpinteria, 6G11 1:50 CDX2 Dako, Carpinteria, DAKCDX2 Ready-to-use IMP3 Dako, Carpinteria, :100 Napsin A Cell Marque, Rocklin, Polyclonal Ready-to-use Abbreviations: 34bE12, high molecular-weight cytokeratin; CDX2, caudal type homeobox 2; HMK, high molecular-weight keratin; HRP-DAB, horseradish peroxidase 3,3 0 diaminobenzidine tetrahydrochloride dihydrate; IMP3, insulin-like growth factor II MRNA-binding protein 3; N- cadherin, neural cadherin; TRS, target retrieval system ; TTF-1, thyroid transcription factor-1. (N-cadherin), caudal type homeobox 2 (CDX2), and insulinlike growth factor II mrna-binding protein 3 (IMP3) have not been used in larger studies comparing these 2 entities. We evaluated the efficacy of this panel in discriminating pulmonary from esophageal adenocarcinoma. MATERIALS AND METHODS 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-lm-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 608C. 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, ) 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 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 4þ as 0, no staining; 1þ, 1% to 25%; 2þ, 26% to 50%; 3þ, 51% to 75%; and 4þ, 76% to 100%; Intensity of staining by TTF-1 and CDX2 were graded using the following scale: 1þ, weak; 2þ, moderate; and 3þ, 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. RESULTS Percentage of Positive Cells 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 34bE12, which were not significantly different from esophageal sites. Staining Intensity 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 34bE12 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 ¼ 22) versus 54% (n ¼ 13), 83% (n ¼ 20) versus 50% (n ¼ 12), 46% (n ¼ 11) versus 17% (n ¼ 4), and 92% (n ¼ 22) versus 75% (n ¼ 18), respectively. Composite Score 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 34bE12. 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. 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3 Table 2. Cut-Off Points 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. Panel Proposal Using the cutoff values in Table 4 in multivariable logisticregression 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. CONCLUSION 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 Staining Intensity Cases, No (%) Stain Intensity Grade Esophagus, n ¼ 24 Lung, n ¼ 24 P Value TTF-1 Negative 3 (13) 2 (8),.001 1þ 8 (33) 0 (0) 2þ 9 (38) 3 (13) 3þ 4 (17) 19 (79) Napsin A Negative 5 (21) 2 (8),.001 1þ 7 (29) 2 (8) 2þ 8 (33) 0 (0) 3þ 4 (17) 20 (83) CDX2 Negative 2 (8) 19 (79),.001 1þ 1 (4) 0 (0) 2þ 5 (21) 1 (4) 3þ 16 (67) 4 (17) IMP3 Negative 1 (4) 15 (63),.001 1þ 5 () 2 (8) 2þ 4 (17) 3 (13) 3þ 14 (58) 4 (17) N-cadherin Negative 24 (100) 14 (58),.001 1þ 0 (0) 2 (8) 2þ 0 (0) 3 (13) 3þ 0 (0) 4 (17) p63 Negative 20 (83) 12 (50).04 1þ 0 (0) 1 (4) 2þ 0 (0) 3 (13) 3þ 4 (17) 8 (33) 34bE12 Negative 2 (8) 1 (4).44 1þ 4 (17) 1 (4) 2þ 4 (17) 3 (13) 3þ 14 (58) 19 (79) Abbreviations: 34bE12, high molecular-weight cytokeratin; CDX2, caudal type homeobox 2; IMP3, insulin-like growth factor II MRNA-binding protein 3; N-cadherin, neural cadherin; p63, transformation-related protein 63; TTF-1, thyroid transcription factor-1. 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 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 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 study 16 found IMP3 to be a highly sensitive and specific biomarker for the diagnosis of invasive esophageal adenocarcinoma and high-grade dysplasia, 1096 Arch Pathol Lab Med Vol 137, August 2013 TTF-1 and Napsin A Utility in Esophageal Tumors Aulakh et al

4 Table 3. Composite Score Cases, No. (%) Stain Composite Score Esophagus, n ¼ 24 Lung, n ¼ 24 P Value TTF-1 Negative 3 (13) 2 (8),.001 2þ 2 (8) 0 (0) 3þ 2 (8) 0 (0) 5þ 6 (25) 0 (0) 6þ 4 (17) 3 (13) 7þ 4 (17) 17 (71) Napsin A Negative 5 (21) 2 (8),.001 2þ 2 (8) 2 (8) 3þ 2 (8) 0 (0) 4þ 4 (17) 1 (4) 5þ 3 (13) 0 (0) 6þ 5 (21) 1 (4) 7þ 3 (13) 18 (75) CDX2 Negative 2 (8) 19 (79),.001 2þ 1 (4) 0 (0) 3þ 1 (4) 0 (0) 5þ 2 (8) 2 (8) 6þ 2 (8) 0 (0) 7þ 13 (54) 1 (4) IMP3 Negative 1 (4) 15 (63),.001 2þ 0 (0) 1 (4) 3þ 0 (0) 3 (13) 4þ 3 (13) 1 (4) 5þ 5 (21) 0 (0) 6þ 5 (21) 2 (8) 7þ 10 (42) 2 (8) N-cadherin Negative 24 (100) 14 (58),.001 2þ 0 (0) 2 (8) 3þ 0 (0) 2 (8) 4þ 0 (0) 2 (8) 5þ 0 (0) 2 (8) 6þ 0 (0) 2 (8) 7þ 0 (0) 2 (8) p63 Negative 20 (83) 12 (50).08 2þ 0 (0) 1 (4) 3þ 0 (0) 2 (8) 5þ 1 (4) 3 (13) 6þ 0 (0) 3 (13) 7þ 0 (0) 1 (4) 34bE12 Negative 2 (8) 1 (4).47 2þ 2 (8) 0 (0) 3þ 1 (4) 0 (0) 4þ 2 (8) 3 (13) 5þ 5 (21) 2 (8) 6þ 3 (13) 5 (21) 7þ 9 (38) 13 (54) Abbreviations: 34bE12, high molecular-weight cytokeratin; CDX2, caudal type homeobox 2; IMP3, insulin-like growth factor II MRNA-binding protein 3; N-cadherin, neural cadherin; p63, transformation-related protein 63; TTF-1, thyroid transcription factor-1. Table 4. Composite Score With Cutoff Points Univariate Logistic Regression Analysis Cutoff Point Multivariate Logistic Regression Analysis Stain Odds Ratio P Value for Esophagus Odds Ratio P Value TTF Napsin A CDX , IMP , ,.001 N-cadherin 0.386, p bE Abbreviations: 34bE12, high molecular-weight cytokeratin; CDX2, caudal type homeobox 2; IMP3, insulin-like growth factor II MRNA-binding protein 3; N-cadherin, neural cadherin; p63, transformation-related protein 63; TTF-1, thyroid transcription factor-1. Arch Pathol Lab Med Vol 137, August 2013 TTF-1 and Napsin A Utility in Esophageal Tumors Aulakh et al 1097

5 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 34bE12 in either esophagus or lung primaries. Therefore, 34bE12 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. References 1. Mizobuchi S, Tachimori Y, Kato H, Watanabe H, Nakanishi Y, Ochiai A. Metastatic esophageal tumors from distant primary lesions: report of three esophagectomies and study of 1835 autopsy cases. Jpn J Clin Oncol. 1997; 27(6) Bejarano PA, Baughman RP, Biddinger PW, et al. Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas. Mod Pathol. 1996;9(4): Whithaus K, Fukuoka J, Prihoda TJ, Jagirdar J. Evaluation of napsin a, cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma versus squamous cell carcinoma of the lung. Arch Pathol Lab Med. 2012;136(2): Minoo P, Hamdan H, Bu D, Warburton D, Stepanik P, delemos R. TTF-1 regulates lung epithelial morphogenesis. Dev Biol. 1995;172(2): Crisera CA, Connelly PR, Marmureanu AR, et al. TTF-1 and HNF-3b in the developing tracheoesophageal fistula: further evidence for the respiratory origin of the distal esophagus. J Pediatr Surg. 1999;34(9): Crisera CA, Maldonado TS, Kadison AS, Li M, Longaker MT, Gittes GK. Patterning of the distal esophagus in esophageal atresia with tracheoesophageal fistula: is thyroid transcription factor 1 a player? J Surg Res. 2000; 92(2): Spilde TL, Bhatia AM, Miller KA, et al. Thyroid transcription factor-1 expression in the human neonatal tracheoesophageal fistula. J Pediatr Surg. 2002; 37(7): Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J. Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med. 2012; 136(2): Odze RD, Goldblum JR. Surgical Pathology of the GI Tract, Liver, Biliary Tract and Pancreas. 2nd ed. Philadelphia, PA: Elsevier; Robens J, Goldstein L, Gown AM, Schnitt SJ. Thyroid transcription factor-1 expression in breast carcinomas. Am J Surg Pathol. 2010;34(12): Fernández-Aceñero MJ, Córdova S, Santonja C. Aberrant TTF-1 expression in papillary high-grade urothelial neoplasm: case report and literature review. Rom J Morphol Embryol. 2011;52(1): Xu B, Thong N, Tan D, Khoury T. Expression of thyroid transcription factor- 1 in colorectal carcinoma. Appl Immunohistochem Mol Morphol. 2010;18(3): Mazziotta RM, Borczuk AC, Powell CA, Mansukhani M. CDX2 immunostaining as a gastrointestinal marker: expression in lung carcinomas is a potential pitfall. Appl Immunohistochem Mol Morphol. 2005;13(1): Rossi G, Cavazza A. CDX2 expression and lung cancer. Appl Immunohistochem Mol Morphol. 2006;14(2): Strickland-Marmol LB, Khoor A, Livingston SK, Rojiani A. Utility of tissuespecific transcription factors thyroid transcription factor 1 and Cdx2 in determining the primary site of metastatic adenocarcinomas to the brain. Arch Pathology Lab Med. 2007;131(11): Lu D, Vohra P, Chu PG, Woda B, Rock KL, Jiang Z. An oncofetal protein IMP3: a new molecular marker for the detection of esophageal adenocarcinoma and high-grade dysplasia. Am J Surg Pathol. 2009;33(4): Kotb AM, Hierholzer A, Kemler R. Replacement of E-cadherin by N- cadherin in the mammary gland leads to fibrocystic changes and tumor formation. Breast Cancer Res. 2011;13(5):R104. doi: /bcr Arch Pathol Lab Med Vol 137, August 2013 TTF-1 and Napsin A Utility in Esophageal Tumors Aulakh et al