American Journal of Surgical Pathology:
Pulmonary Sclerosing Hemangioma Consistently Expresses Thyroid Transcription Factor-1 (TTF-1): A New Clue to Its Histogenesis
Chan, Alexander C. L. M.B.B.S., F.R.C.P.A.; Chan, John K. C. M.B.B.S., F.R.C.Path.
From the Department of Pathology, Queen Elizabeth Hospital, Hong Kong.
Address correspondence and reprint requests to Alexander C. L. Chan, MBBS, FRCPA, Department of Pathology, Queen Elizabeth Hospital, 30 Gascoigne Road, Kowloon, Hong Kong; e-mail[colon] chancl[commat]ha.org.hk
The histogenesis of pulmonary sclerosing hemangioma has remained controversial despite extensive studies by many investigators. The availability of an antibody to thyroid transcription factor-1 (TTF-1), which is expressed in type II pneumocytes and Clara cells, has prompted us to readdress this issue. Sixteen cases were immunostained with a panel of antibodies including TTF-1. The patients were predominantly women with an age range of 30 to 73 years (mean, 52 yrs). All tumors were solitary. The single male patient showed regional lymph node metastases, an unusual occurrence reported only once in the literature. All cases exhibited the classic histologic features, with variegated patterns. TTF-1 expression was observed in both the surface lining cells and the pale polygonal cells. The surface lining cells were epithelial membrane antigen (EMA)[plus] cytokeratin[plus] surfactant apoprotein A[plus], whereas the polygonal cells were EMA[plus] cytokeratin-surfactant apoprotein A-. The neuroendocrine markers synaptophysin and chromogranin were both negative. The metastatic deposits in the lymph nodes comprised only polygonal cells and exhibited an EMA[plus] cytokeratin-surfactant apoprotein A- TTF-1[plus] immunophenotype. These results suggest that pulmonary sclerosing hemangioma is an epithelial neoplasm derived from primitive respiratory epithelium or incompletely differentiated type II pneumocyte or Clara cell.
Sclerosing hemangioma of the lung is an uncommon benign tumor first described by Liebow and Hubell in 1956. 20 Histologically, there is a mixture of solid, sclerotic, papillary, and hemorrhagic patterns. Two populations of cells can be identified, solid-growing bland polygonal cells with abundant pale cytoplasm and cuboidal lining cells covering the papillary structures.
Despite its characteristic gross and histologic appearance, the histogenesis of this tumor has remained controversial. The tumor has variously been considered to show endothelial, mesothelial, epithelial, and recently, neuroendocrine differentiation. 10,13,20,33,34 While the pale polygonal cells are widely accepted to represent the neoplastic component, whether the surface cuboidal cells represent part of the neoplasm or entrapped pneumocytes is still under debate. Although the polygonal cells express epithelial membrane antigen (EMA), cytokeratin expression is usually lacking according to most studies. 11,26,33 Thus, support for an epithelial nature of pulmonary sclerosing hemangioma is less than conclusive.
Thyroid transcription factor-1 (TTF-1) is a 38 kDa nuclear protein member of the NKx2 homeodomain transcription factor family. 32 It is expressed in the thyroid, lung, and diencephalon of the brain. 16 In the lung, it binds to and regulates the surfactant A, B, C, and Clara cell secretory protein genes. 2,3,15,35 Its expression can be detected immunohistochemically in the columnar nonciliated cells of the fetal lung at as early as 11 weeks of gestation. 28 In adults, it is expressed only in type II pneumocytes and Clara cells. 16 TTF-1 has been detected in different types of lung tumors, especially alveolar adenoma (100[percnt]), adenocarcinoma (72.5[percnt]), small cell carcinoma (94.1[percnt]), and atypical carcinoid (100[percnt]). 9,14,25 It is considered to be superior to surfactant as a marker for lung carcinoma because of its higher sensitivity. 14,16 It is not expressed in mesotheliomas, and is hence helpful for distinction between pulmonary adenocarcinoma and mesothelioma. 16 It is seldom expressed in tumors of other organs, with the exception of thyroid tumors, as expected from its distribution in normal tissues, and some nonpulmonary small cell carcinomas. 1,25 Because of the restricted expression of TTF-1 in normal tissues and tumors, immunostaining for this marker can potentially shed light on the nature of pulmonary sclerosing hemangioma.
MATERIALS AND METHODS
All cases of pulmonary sclerosing hemangioma diagnosed in the Department of Pathology, Queen Elizabeth Hospital, Hong Kong, from 1991 to 2000 were retrieved. Sixteen cases were identified (see Table 1), and all were surgical resection specimens. The hematoxylin and eosin-stained slides were reviewed to reconfirm the diagnosis. Immunohistochemical staining was performed on paraffin sections using labeled streptavidin-biotin peroxidase technique with an automated immunostainer (Ventana, Tucson, AZ, USA). The panel of antibodies included anti-TTF-1 (dilution 1[colon]10, clone 8G7G3/1, NeoMarkers, Union City, CA, USA), anti-pancytokeratin (dilution 1[colon]50, clone MNF116, Dako, Glostrup, Denmark), anti-EMA (dilution 1[colon]10, clone Mc5, Ventana), anti-chromogranin (dilution[colon] 1[colon]100, clone LK2H10, BioGenex, San Ramon, CA, USA), anti-synaptophysin (dilution[colon] 1[colon]50, polyclonal, Dako), and anti-surfactant apoprotein A (dilution[colon] 1[colon]150, clone PE10, Dako). Antigen retrieval was effected by pressure cooking in EDTA buffer, pH 8.0 for 2.5 minutes. The amplification kit (Ventana) was applied for TTF-1. The endogenous biotin blocking kit (Ventana) was used for chromogranin and synaptophysin immunostaining to minimize problems in interpretation. Sausage block containing miscellaneous tissues such as normal thyroid, lung tissue, and colonic tissue was mounted on every slide to provide positive controls. A case was regarded as positive for TTF-1 if more than 10[percnt] of the specific cell population showed nuclear staining.
Among the 16 cases, there was marked female predominance, with a female to male ratio of 15[colon]1. The ages ranged from 30 to 73 years, with a median of 53 and a mean of 52. Ten tumors occurred in the lower lobes, five in the right middle lobe, and one in the right upper lobe. All were solitary lesions. The tumor size ranged from 1.0 to 8.0 cm, with a median of 2.0 cm and a mean of 2.4 cm. All these findings were comparable to those reported in the literature. 5[ndash]7 The largest tumor occurred in the single male patient, whose regional lymph nodes showed metastases. This tumor was already discovered 31 years before the operation, when the patient was 17 years old, but was not removed because it was considered benign radiologically and clinically.
All cases showed the typical histologic appearances of sclerosing hemangioma, with variable proportions of solid, papillary, hemorrhagic, and sclerotic patterns (Fig. 1). The tumors consisted of solid sheets and aggregates of pale polygonal cells (Fig. 2) punctated by papillae and clefts lined by cuboidal or columnar cells. In the single case with hilar lymph node metastases, one lymph node contained two foci measuring 2 mm and 0.7 mm, respectively, and another contained a 0.5-mm focus. The metastatic deposits consisted of a monotonous population of pale polygonal cells associated with a rich vascular network (Fig. 3). The histologic appearance of the primary tumor was indistinguishable from other nonmetastasizing cases.
Immunohistochemically, all cases showed similar results. EMA staining was observed in both the pale polygonal cells (10[percnt][ndash]95[percnt]) and the surface cuboidal cells (20[percnt][ndash]100[percnt]) in a membranous and cytoplasmic pattern, whereas cytokeratin expression was limited to some of the cuboidal cells (10[percnt][ndash]100[percnt]) lining the papillae and clefts (Fig. 4). All the tumors expressed TTF-1 in both the polygonal and surface cuboidal cells (Fig. 4). In some cases, TTF-1 staining was weaker in the center of the tissue block, attributable to delayed fixation causing degradation of the antigen. Although many polygonal cells had an EMA[plus] TTF-1[plus] profile, some showed an EMA-TTF-1[plus] pattern. Thus, TTF-1 appeared to be a more sensitive marker in detecting the polygonal cells than EMA. The population of cells showing surfactant apoprotein A staining paralleled that showing cytokeratin expression and the pattern of staining was granular. The polygonal cells were negative for surfactant apoprotein A. In the case with lymph node metastases, the metastatic tumor cells expressed TTF-1 and EMA, but not cytokeratin or surfactant apoprotein A (Fig. 3B). All tumors were negative for chromogranin and synaptophysin. The normal neuroendocrine cells in the adjacent nonneoplastic bronchi served as internal positive controls.
This study on 16 cases of pulmonary sclerosing hemangioma shows that cytokeratin expression is limited to the surface lining cells, whereas EMA is expressed in both the polygonal cells and the lining cells. These findings are in keeping with those of Haimoto et al., 11 Xu et al., 33 and Rodriguez-Soto et al., 26 but in contrast to those of Leong et al., 19 Yousem et al., 34 and Nagata et al. 21 Based on the more recent studies applying effective antigen retrieval techniques (current study and that of Rodriguez-Soto et al. 26), it appears that the polygonal cells are usually cytokeratin-negative, and if positive, are usually weak and in a minority of cells. This peculiar cytokeratin-EMA[plus] immunophenotype can be helpful in confirming a diagnosis of sclerosing hemangioma in equivocal cases, especially in biopsy materials when the characteristic variegated pattern is not evident.
The debate on the nature of sclerosing hemangioma was recently rekindled by the study of Xu et al., who reported that the polygonal cells expressed a number of neuroendocrine markers, including chromogranin A, neuron-specific enolase, synaptophysin, adrenocorticotropic hormone, growth hormone, calcitonin, and gastrin; they also interpreted the dense core granules identified ultrastructurally as neurosecretory granules. 33 Based on these findings, the authors suggested renaming the tumor [ldquo]benign neuroendocrine tumor of the lung.[rdquo]33 However, these results cannot be reproduced by two other groups 23,26 and our current study. Thus, we find no support for the presence of neuroendocrine differentiation in this neoplasm. We suspect that the observed staining reported by Xu et al. might be the result of endogenous biotin activity or nonspecific staining. 4
Study for TTF-1 expression offers a unique opportunity to assess the line of differentiation in sclerosing hemangioma. The identification of nuclear expression of TTF-1 in both the polygonal and cuboidal cells raises the possibility of derivation from or differentiation toward type II pneumocytes and Clara cells. Yousem et al. 34 have demonstrated Clara cell antigen and surfactant apoprotein in the polygonal cells of sclerosing hemangioma. Nagata et al. 22 have also shown that some polygonal cells expressed surfactant apoprotein and secretory component. On the other hand, our study and that of Leong et al. 19 could not identify surfactant expression in the polygonal cells. These findings suggest that the reported cytoplasmic staining for surfactant in the polygonal cells might have resulted from absorption of surfactant from the surrounding lung tissues. Because only the surface lining cells expressed surfactant, Leong et al. suggested that the lining cells were entrapped elements instead of neoplastic cells. 18 Nonetheless, there is now some morphologic and molecular evidence that both the polygonal and cuboidal cells are neoplastic, helping to end this debate. In an example of invasive sclerosing hemangioma, the invasive component within the bronchial lumen contains papillary component with surfactant expression, indicating that the lining cells are also neoplastic. 23 Niho et al. have shown by microdissection that both the lining cells and the polygonal cells (their [ldquo]pale cells[rdquo]) belonged to the same clone using restriction fragment length polymorphism analysis on the X-chromosome-linked human androgen receptor (HUMARA) gene or phosphoglycerate kinase (PGK) gene, and thus concluded that the two populations represented variable differentiation from a progenitor cell. 24 However, the lack of cytokeratin expression in the polygonal cells of sclerosing hemangioma remains a mystery, because normal and neoplastic type II pneumocytes and Clara cells (for example, in bronchioloalveolar carcinoma) almost consistently express cytokeratin. Can the polygonal cells recapitulate a third type of yet unrecognized TTF-1-positive and cytokeratin-negative subepithelial cell in the lung that may be the progenitor of normal Clara cells and type II pneumocytes? We have performed immunostaining for TTF-1 on fetal and normal lungs, but have failed to identify any TTF-1-positive cells other than those that line the lumens. Thus, these TTF-1[plus] polygonal cells appear to represent neoplastic cells with no normal counterpart. Nonetheless, their TTF-1 immunoreactivity provides a strong link to the epithelial cells of the lung despite lack of cytokeratin expression. Although TTF-1 is also commonly expressed in pulmonary neuroendocrine tumors, 9,25 there is no immunohistochemical evidence of neuroendocrine differentiation in sclerosing hemangioma. We therefore think the polygonal cells in sclerosing hemangioma may have originated from primitive respiratory epithelium or may represent incompletely differentiated pneumocytes or Clara cells, and they can mature in areas into cuboidal cells achieving the full phenotype of type II pneumocytes or Clara cells.
Another significant finding of this series of sclerosing hemangioma is the documentation of metastasis in two regional lymph nodes in one patient with a large tumor (8 cm). This phenomenon has only been previously documented in one case in the literature by Tanaka et al., 29 and that case had also been included in the series by Spencer and Nambu. 26 Interestingly, similar to our case, the patient was also a male with a large tumor (5 cm), and the metastatic deposit was only identified in the hilar lymph node microscopically. Although focal papillary area was observed in the lymph node metastasis in the case by Tanaka et al., 29 only the polygonal cell component has metastasized in our case. The distinctive cytokeratin-EMA[plus] TTF-1[plus] immunophenotypic profile provides strong support for the interpretation of the nodal deposits as metastatic sclerosing hemangioma, and it helps to exclude the possibility of metastasis from an occult carcinoma. Unfortunately, postoperative follow-up information was not available for both cases (our case was recent), and thus the significance of regional nodal metastasis remains to be determined. Nonetheless, the identification of metastasis in rare cases of sclerosing hemangioma does not necessarily put this group of tumors in the potentially malignant category, because other benign tumors have also been reported to metastasize, for example, metastasizing pleomorphic adenoma, 31 giant cell tumor of bone, 30 and chondroblastoma. 12
Devouassoux-Shisheboran et al 8 have recently published similar findings, with TTF-1 expression identified in both the surface cuboidal cells and the pale polygonal cells in pulmonary sclerosing hemangioma. Surfactant proteins A and B as well as Clara cell antigen were consistently negative in the pale polygonal cells in their cases. They suggested an origin of the tumor cells from primitive respiratory epithelium. Their series also included a single case with peribronchial lymph node metastasis.
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Thyroid transcription factor-1; TTF-1; Epithelial membrane antigen; Lung neoplasm; Sclerosing hemangioma; Immunohistochemistry
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