Expression Patterns of Markers for Type II Pneumocytes in Pulmonary Sclerosing Hemangiomas and Fetal Lung Tissues

Pulmonary sclerosing hemangioma is an uncommon epithelial tumor with a predilection for middle-aged women. Most of the cases follow a benign clinical course, but a few regional lymph node metastases have been reported.1-4 According to the World Health Organization classification revised in 2001,5 sclerosing hemangioma is a tumor of uncertain type with a distinctive constellation of histologie findings, exhibiting the following histologically characteristic architectural patterns: papillary, solid, sclerotic, hemorrhagic, and mixed. This tumor contains 2 types of tumor cells, pale polygonal cells and cuboidal cells lining the papillary structure; however, it has been controversial which type is neoplastic. The pale polygonal cells were initially regarded as the neoplastic component of sclerosing hemangioma, whereas the cuboidal cells were believed to be entrapped alveolar epithelial cells. However, in a recent study,6 the clonal nature of both of these 2 cell types was demonstrated using X-chromosome inactivation analysis, suggesting that both cell types are neoplastic. Immunohistochemical and ultrastructural analyses have suggested that these neoplastic cells may originate from epithelial, neuroendocrine, mesenchymal, mesothelial, and endothelial differentiation.7-13 Recently, it has been suggested in 2 publications that the pale polygonal cells of sclerosing hemangioma could be derived from primitive undifferentiated respiratory epithelium and then differentiate toward type II pneumocytes or Clara cells. Devouassoux-Shisheboran et al2 observed the expression of thyroid transcription factor 1 (TTRl), cytokeratin, surfactant A and B, Clara cell antigen, and epithelial membrane antigen (EMA) in surface lining cells, but lack of expression of cytokeratin, surfactant A and B, and Clara cell antigens in pale polygonal cells. They concluded that the pale polygonal cells of sclerosing hemangioma recapitulate primitive and undifferentiated respiratory epithelium. Chan et al1 also suggested sclerosing hemangioma is an epithelial neoplasm derived from primitive respiratory epithelium or incompletely differentiated type II pneumocyte or Clara cells. Nonetheless, the precise differentiation lineage of neoplastic cells in this rare benign tumor remains somewhat questionable.

To address this issue, we studied 15 cases of sclerosing hemangioma and 15 specimens of fetal lung tissue by immunohistochemistry using antibodies against MUCl, CD44v6, TTFl, and Thomsen-Friedenreich (TF) antigen. In this study, 2 types of tumor cells differentially expressed these antigens, which were expressed in developing type II pneumocytes of fetal lung as well as mature type II pneumocytes of adult lung. These findings suggested that 2 types of cells in sclerosing hemangioma may have been derived from a common precursor and may divergently differentiate toward the type II pneumocyte during tumorigenesis.

MATERIALS AND METHODS

Fifteen patients with sclerosing hemangioma undergoing surgical resection between 1993 and 2002 were included in this retrospective study. All patients were examined and studied at the Department of Pathology, Seoul National University Hospital, Seoul, Korea. Fetal lung tissues obtained from the autopsy files at Seoul National University Children's Hospital included pseudoglandular (6 cases), canalicular (3 cases), saccular (4 cases), and alveolar stages (2 cases). Slides were reviewed and reevaluated for histologie features by 2 pathologists (S.H.Y., D.H.C.).

Immunoperoxidase studies were performed on 4-µm-thick, formalin-fixed, paraffin-embedded tissue sections using the standard avidin-biotin complex technique. We immunostained specimens from 15 cases of sclerosing hemangioma and 15 cases of fetal lung tissue using anti-MUCl (dilution 1:100, clone Ma 695, Novocastra, Newcastle upon Tyne, United Kingdom), anti-CD44v6 (dilution 1:400, clone VFF7, Serotec, Kidlington, United Kingdom), anti-TF (dilution 1:100, clone HB-Tl, DakoCytomation Denmark A/S, Glostrup, Denmark), and anti-TTF-1 (dilution 1: 100, clone 8G7G3, DakoCytomation, Carpinteria, Calif). In addition, we immunostained 15 cases of sclerosing hemangioma with a panel of antibodies including anti-cytokeratin (dilution 1: 300, clone MNF 116, DakoCytomation), anti-EMA (dilution 1:100, clone E29, DakoCytomation), anti-synaptophysin (dilution 1:50, polyclonal, DakoCytomation), anti-CD56 (dilution 1:100, clone 123C3, Zymed Laboratories, San Francisco, Calif), anti-estrogen receptor (dilution 1:80, clone 1D5, DakoCytomation), and antiprogesterone receptor (dilution 1:80, clone 1A6, DakoCytomation). In the treatment of anti-TF antibody, tissues were pretreated with sialidase (from Arthrobacter ureafaciens, Nakarai Chemicals, Kyoto, Japan; 1 unit/mL in 0.05 M phosphate buffer, pH 7.0) at 37°C for 12 hours. In immunostaining with anti-CD44v6 antibody, horseradish peroxidase-conjugated streptavidin (DiNonA, Seoul, Korea) was directly applied to tissue sections and incubated for 20 minutes at room temperature before primary antibodies were applied. The extent of immunoreactivity was recorded for both surface lining cells and pale polygonal cells as positive (=10% with cytoplasmic staining or nuclear staining) or negative (<10%). Negative controls in which the primary antibody was omitted and positive controls on tissues with known positive immunoreaction for each antibody were used.

RESULTS

All 15 patients were women and ranged in age from 26 to 70 years (mean, 51 years). Six tumors were located in the right middle lobe, 5 in the right lower lobe, 2 in the right upper lobe, 1 in the left lower lobe, and 1 in the left upper lobe. The size of the tumors ranged from 1.0 to 6.5 cm with an average of 2.9 cm. Histologically, all cases showed varying proportions of solid (Figure 1, A), papillary (Figure 1, B), sclerotic (Figure 1, C), and herrtorrhagic patterns (Figure 1, D). The tumors were composed of the typical 2 types of cells, pale polygonal cells and surface lining cells. Solid areas consisted of pale polygonal cells with well-demarcated cytoplasmic borders and round to oval nuclei. These pale polygonal cells were also detected in the center of a papillary pattern, in the vicinity of a hemorrhagic pattern, and in the cellular area around sclerotic foci. Surface lining cells, which are cuboidal or columnar with large prominent nuclei, were noted mainly in the papillary area. In solid areas, the foci of surface lining cells were intermingled with sheets of pale polygonal cells.

To clarify the precise lineage of differentiation for neoplastic cells, we performed immunohistochemical analysis of sclerosing hemangioma using various epithelial, mesenchymal, neuroendocrine, and type II pneumocyte markers. The results of this analysis using sclerosing hemangioma and fetal lung specimens are summarized in Table 1 and Table 2, respectively. As previously reported, staining for EMA was shown on both pale polygonal cells and surface lining cells in all 15 cases, whereas expression of pancytokeratin was not detected on pale polygonal cells, but was present on surface lining cells. Nuclear staining for estrogen receptor and progesterone receptor and membrane staining for CD56 and synaptophysin were not observed in either of the cell types in sclerosing hemangioma. The expression patterns of type II pneumocyte markers, such as MUCl, CD44v6, TTF-I, and TF antigen, were examined in pale polygonal and surface lining cells of both sclerosing hemangiomas and fetal lung tissues. In the fetal lung specimens, MUCl and TTF-I were expressed throughout all stages of airway and bronchial epithelium. Thomsen-Friedenreich antigen was positive in bronchial buds of the pseudoglandular stage, in respiratory bronchioles and alveolar ducts of the canalicular stage, and in terminal sac type II cells of the saccular and alveolar stages. CD44v6 was highly expressed on type II pneumocytes of the saccular and alveolar stages, indicating that expression of CD44v6 is mainly expressed in developing type II pneumocytes during the late stage of pulmonary development and in mature type II pneumocytes after birth. In addition, none of markers tested for type II pneumocytes were expressed by Clara cells in lung parenchyma. In 15 cases of sclerosing hemangioma, the expression of MUCl was observed in both the surface lining cells and the pale polygonal cells (Figure 2, A). All tumors expressed nuclear staining with TTF-I in both pale polygonal and surface lining cells (Figure 2, B). In 11 of 15 cases of sclerosing hemangioma, surface epithelial cells showed positive expression for TF antigen on apical surfaces (Figure 2, C), whereas none of the cases demonstrated positive staining of pale polygonal cells with anti-TF antibody. CD44v6 was expressed on surface lining cells in a basolateral membranous pattern, but not on pale polygonal cells (Figure 2, D).

COMMENT

In the present study, we performed immunohistochemical analysis using antibodies against MUClx TF antigen, TTF-1, and CD44v6 as lineage markers of type II pneumocyte differentiation. MUCl, one of the mucin families of glycoprotein, is highly expressed in normal type II pneumocytes and lesions derived from type II pneumocytes, such as type II cell hyperplasia, dysplastic type II cells, and apical alveolar hyperplasia.14,15 Thomsen-Friedenreich antigen is a cryptic glycoprotein masked by some carbohydrates in normal tissue.16 Immunostaining with a monoclonal antibody against TF antigen after removal of sialic acids results in specific staining of type II pneumocytes.17-19 CD44 is a cell-surface adhesion molecule mediating both cell to cell and cell to matrix interaction.20 In our study, TTF-I and MUCl were expressed by most type II pneumocytes, but only in tiny focal areas of bronchial epithelium, and TF antigen was detected on type II pneumocytes, but not on bronchial epithelial cells. Based on these results, we believe that TTF-I, MUCl, and TF antigen were useful markers for type II pneumocytes, although TTF-I and MUCl were expressed in a focal area of bronchial-bronchiolar epithelium.

Among the markers for type II pneumocytes tested, TTF-I and MUCl were expressed by respiratory epithelium through all developmental stages of fetal lung. CD44v6 and TF antigens were expressed on developing type II pneumocytes of saccular and alveolar stages of the fetal lung and on mature type II pneumocytes after birth. Kasper et al21 documented that CD44v6 was found only during the saccular stage of human fetal lung tissue. In the 15 cases of sclerosing hemangioma tested, the surface lining cells expressed EMA, cytokeratin, MUCl, TF antigen, TTF-I, and CD44v6, whereas pale polygonal cells expressed EMA, TTF-I, and MUCl, suggesting that surface lining cells are fully differentiated toward the type II pneumocyte, whereas pale polygonal cells remain at the primitive or late stage of development in respiratory epithelium. Thus, it may be feasible that surface lining cells derive from the type II pneumocyte, and pale polygonal cells originate from primitive respiratory epithelium. However, Niho et al6 demonstrated the monoclonality of pale polygonal and surface lining cells using a microdis section technique and X-chromosome methylation patterns, indicating that 2 types of cells of sclerosing hemangioma have the same origin. Based on these results, it has been suggested in previously published reports that the 2 types of cells derive from common precursor cells, such as primitive respiratory epithelium and type II pneumocytes. These results led us to hypothesize that common precursor cells of sclerosing hemangioma should be divergently differentiated into mature respiratory epithelium during tumorigenesis. Thus, one group of tumor cells is differentiated into type II pneumocytes, while the other group of cells becomes less differentiated and remains at the primitive stage of respiratory epithelium. Expression of CD44v6, MUCl, and TF antigen was not evident on Clara cells, indicating that CD44v6 and TF antigens are excellent markers for developing and mature type II pneumocytes, but not for Clara cells. Furthermore, the ultrastructural findings of sclerosing hemangioma supported that surface lining cells differentiate into type II pneumocytes.22,23 Thus, the expression of CD44v6, MUCl, and TF antigen on surface lining cells of sclerosing hemangioma indicated that these cells are fully differentiated into type II pneumocytes rather than Clara cells.

Our findings suggest that sclerosing hemangioma is derived from common precursor cells that divergently differentiate into type II pneumocytes rather than Clara cells during tumorigenesis (Figure 3). In addition, our immunohistochemistry results will be helpful in the differential diagnosis of sclerosing hemangioma, as well as in the diagnosis of unusual presentations of sclerosing hemangioma, including lymph node metastasis, in endobronchial, pleural, and mediastinal locations.2

In summary, the immunohistochemical study of sclerosing hemangioma using antibodies against type II pneumocytes provided a novel concept for the histogenesis of sclerosing hemangioma and was a useful diagnostic tool for this benign pulmonary neoplasm.

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