Expression of thyroid transcription factor-1 and other markers in sclerosing hemangioma of the lung.

First described by Liebow and Hubbell in 1956, (1) sclerosing hemangioma (SH) of the lung is a generally benign tumor of uncertain origin with a characteristic gross and histologic appearance. Typically, it presents as a circumscribed round or oval mass averaging 2.8 cm. (1-3) Histologically, the tumor may display solid, papillary, hemorrhagic, and sclerotic areas. In more than 90% of the cases, at least 3 of these patterns are present (3) and 2 cell types can be identified, namely, the pale cells in the solid areas and the cuboidal cells lining the papillary structures. Despite the implication of its descriptive name, immunohistochemical and ultrastructural studies have shown that the tumor cells of SH are of epithelial rather than endothelial nature. (4-11) However, most markers are expressed only focally and weakly in the pale cells and erratically in some or all of the cuboidal cells, suggesting that some of the latter represent entrapped metaplastic alveolar epithelial elements. Furthermore, the nature of these epithelial cells, and specifically their relationship to the pulmonary epithelia, is unclear. Interestingly, the clonal nature of both cell types was demonstrated in a recent study using X-chromosome inactivation analysis. (12)

Thyroid transcription factor-1 (TTF-1) is a 38-kd nuclear protein initially identified as a mediator of thyroid-specific gene transcription. (13,14) Subsequently, expression of TTF-1 was detected in lung and ventral forebrain. (15) Immunohistochemical staining for TTF-1 has been used to identify tumors of pulmonary epithelial origin. (16-18)

In this immunohistochemical study, we analyzed the staining pattern of SH for TTF-1 coupled with epithelial (anti-epithelial membrane antigen [EMA], CAM 5.2) and endothelial (CD31) markers, surfactant apoprotein (PE10), and chromogranin to clarify the line of differentiation of this neoplasm.

MATERIALS AND METHODS

Nine cases of histologically typical SH were retrieved from the files of Memorial Sloan-Kettering Cancer Center (New York, NY) and the consultation files of 1 of the authors (J.R.). Clinical information was obtained from the pathology reports. Slides were reviewed and the histologic features were evaluated independently by the 3 authors.

The cases were stained for TTF-1, EMA, low-molecular-weight cytokeratin (CAM 5.2), endothelial cell marker (CD31), surfactant protein B (PE10), and chromogranin A. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections. The sources, dilutions, and treatment conditions for the antibodies are listed in Table 1. Standard 4-[micro]m sections were cut, deparaffinized, and mounted on precoated slides. Sections were treated with 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity and were further treated with heat-induced epitope retrieval (for TTF-1, CD31, EMA, CAM 5.2, and chromogranin). Following pretreatment, sections were incubated for as long as 30 minutes at room temperature with normal horse serum and then with primary antibodies (Table 1) for 12 to 18 hours at 4 [degrees] C. This step was followed by incubation with biotinylated horse anti-mouse immunoglobulin G (IgG, 1: 500; Vector Laboratories, Burlingame, Calif) for 60 minutes at room temperature and with hydrogen peroxide-conjugated streptavidin (1:500 dilution; Dako Corporation, Carpinteria, Calif). The final reaction product was visualized with diaminobenzidine. Sections were counterstained with Harris hematoxylin. Normal lung tissue surrounding the tumor served as a positive internal control. Staining patterns were evaluated separately in the pale cells of solid areas and in the cells lining the papillary structures.

The staining intensity was described as strong (intensity the same or greater than half of the internal positive control) or weak (intensity less than half of the internal positive control). For the epithelial markers (EMA and CAM 5.2), a distinction was made between cytoplasmic and membranous staining. The extent of staining was scored as diffuse (contiguous staining of at least 20% of tumor cells) or focal (contiguous staining in less than 20% of tumor cells).

RESULTS

The patients ranged in age from 28 to 71 years (mean 48 years); 7 were women and 2 were men. The tumor was located in the right lung in 3 cases and in the left lung in 6 cases; there was no predilection to any of the 5 lobes. The cases demonstrated the typical histologic appearance of SH and showed at least 2 different patterns, with solid and papillary being the most common (Figure 1). The solid areas were composed of bland-appearing epithelioid cells with pale cytoplasm, well-defined cytoplasmic borders, and round to oval uniform nuclei with fine chromatin and inconspicuous nucleoli (Figure 2). The cells lining the papillary structures showed different appearances in different regions of the tumors. In many areas, the cells lining the papillae were cuboidal or columnar, with eosinophilic cytoplasm, large prominent nuclei, and inconspicuous or absent nucleoli, closely resembling the pale cells of the solid areas (Figure 2). In the peripheral areas of the tumors, however, there were papillary formations lined by cells with smaller nuclei and minimal cytoplasm arranged in a hobnailed configuration resembling metaplastic type II pneumocytes (Figure 2). Focal foamy changes and cytoplasmic inclusion of hemosiderin were found in these cells. Additional histologic findings were tumor cells with clear cytoplasm, aggregates of foamy histiocytes, mast cells, hemosiderin deposition, calcification, and laminated scroll-like whorls (Figure 3).

[FIGURES 1-3 OMITTED]

A summary of the staining pattern for the various markers tested in the pale cells of the solid areas and the 2 types of cells lining the papillary structures is shown in Table 2. Normal lung tissues surrounding the tumor demonstrated at least focal staining with every antibody, including chromogranin, which showed rare positive cells in the peripheral airways. Stains with PE10, CD31, and chromogranin were not performed in 1 case owing to insufficient tissue.

In all 9 cases, the pale cells of the solid areas demonstrated diffuse nuclear staining for TTF-1, the intensity being the same or slightly weaker than in the type II pneumocytes of the surrounding lung (Figure 4). PE10 showed focal weak staining of the pale cells in 4 cases, focal strong staining in 1 case, and no staining in 3 cases. The pale cells were positive for EMA in all 9 cases, with strong staining in 2 cases and only focal weak staining in 3 cases (Figure 4), whereas reactions for CAM 5.2 were negative in the pale cells in 2 cases and only focally positive in 7 cases. In 4 of the 7 cases with pale cells positive with CAM 5.2, only cytoplasmic staining was identified, and the number of CAM 5.2-positive cells was less than the number of EMA-positive cells.

[FIGURE 4 OMITTED]

The cells lining the papillary structures also showed strong nuclear staining for TTF-1 in all 9 cases. Reactions for PE10, EMA, and CAM 5.2 were positive in some of these cells in all cases, the intensity varying from weak to strong. In particular, the papillary cells resembling the cells of the solid areas showed weaker staining for EMA (Figure 4) and less frequent or absent staining with PE10 and CAM 5.2. The peripheral areas of the tumor with smaller, hobnailed papillary lining cells showed more consistent and intense staining with CAM 5.2 and PE10 (Figure 4), in addition to strong staining for EMA. The number and location of PE10-positive cells appeared to match the number and location of CAM 5.2-positive cells.

Reactions for chromogranin and CD31 were negative for all cell types in all cases.

COMMENT

Since the initial description of SH by Liebow and Hubbell, (1) the nature of the tumor cells has been a source of controversy. The presence of blood-filled spaces lined by flat endothelial-appearing cells and papillary structures lined by cuboidal cells with or without blood in the surrounding spaces initially suggested a vascular neoplasm. (1) However, subsequent electron microscopic and immunohistochemical studies found no evidence of endothelial differentiation (4-8) and demonstrated instead epithelial features. (4-6,9-11,19,20) Ultrastructural findings included dense inclusion bodies, lamellar bodies, and basement membrane, indicating that some of the papillary lining cells were virtually identical to type II pneumocytes.(4,7) The pale cells of the solid areas were characterized by poor development of cytoplasmic organelles, that is, few mitochondria, minimal rough endoplasmic reticulum, scattered free ribosomes, and well to poorly developed desmosomes.(4,7) Immunoelectron microscopic studies showed that some pale cells had a positive reaction with PE10 that was identical to that of type II pneumocytes. (4) The published immunohistochemical studies showed differing staining in the papillary lining cells and the pale cells. The papillary lining cells were consistently EMA positive and showed variable staining for cytokeratins, carcinoembryonic antigen, vimentin, and with PE10. This staining pattern suggests that the papillary structures may represent entrapped alveolar lung tissue lined by metaplastic type II pneumocytes. This notion is further supported by the frequent finding of PE10-positive, laminated, scroll-like whorls in the spaces lined by these cells. On the other hand, the pale cells showed at least some focal EMA positivity in the large majority of cases, and focal cytokeratin, vimentin, and carcinoembryonic antigen staining was also detected in some of the cases. In addition, in 2 recently published studies, TTF-1 positivity was detected in 92% of the pale cells and 97% of the papillary lining cells in one study, (10) and 100% in both cell types in the other study. (11)

Other data cast some doubt on the hypothesis that the papillary lining cells are entrapped nonneoplastic pneumocytes. In a recent study of 6 cases of SH, Niho et al (12) found that both the pale cells and the cuboidal papillary lining cells represented clonal populations. The authors extracted DNA from microdissected methanol-fixed tissues and performed clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene, or the phosphoglycerate kinase gene. Both cell types showed the same monoclonality in all informative cases, leading the authors to conclude that both pale cells and cuboidal cells are derived from the same cell.

Neuroendocrine differentiation was described in a study of 32 cases by Xu et al. (19) Staining for neuron-specific enolase was interpreted as positive in 24 of 24 cases, for chromogranin A in 19 of 22 cases, for adrenocorticotropic hormone and growth hormone in 14 of 15 cases, for calcitonin in 11 of 15 cases, for gastrin in 11 of 14 cases, and for synaptophysin in 6 of 10 cases. The authors concluded that SH is a benign tumor with neuroendocrine differentiation and suggested it be renamed "benign neuroendocrine tumor" of the lung. However, subsequent studies from Japan (20) and the United States (9-11) failed to detect significant neuroendocrine differentiation in a total of 148 cases. Possible reasons for this striking discrepancy include difference in histologic interpretation, variability in staining techniques or reagents, and poor preservation of the tissue.

In our study, we selected a panel of markers to identify possible epithelial (EMA and CAM 5.2), endothelial (CD31), and neuroendocrine (chromogranin) differentiation, including markers for alveolar type II pneumocytes and nonciliated bronchiolar cells (TTF-1 and PE10). We selected CAM 5.2 because of published data demonstrating that low-molecular-weight cytokeratins are one of the most commonly expressed markers in SH and because it is strongly positive in normal lung. (9) Thyroid transcription factor-1 has been identified in human lung as early as 11 weeks of gestation, being localized to the nuclei of epithelial cells of the developing airways. (21,22) Thyroid transcription factor-1 is a potent transcriptional activator of genes specific to the thyroid (thyroglobulin, thyroid peroxidase, thyrotropin receptor) and lung (surfactant proteins A, B, C, and D, and Clara cell secretory protein) and plays a role in the organogenesis of the thyroid gland and the lungs. (20-27) After birth, TTF-1 is selectively expressed in alveolar type II pneumocytes and in a subset of nonciliated cuboidal cells of the conducting airways, (22,27) similar to patterns of distribution of surfactant protein B in developing and pathologic lungs. (22,27)

We found that the only marker uniformly expressed by all cells in all cases was TTF-1. The pale cells of the solid areas were always diffusely and intensely TTF-1 positive and showed consistent positivity of variable intensity for EMA. CAM 5.2 and PE10 were only focally present in the pale cells. These results further support the epithelial nature of the neoplastic component of SH and specifically point toward pulmonary epithelial differentiation. The relatively infrequent expression of CAM 5.2 and PE10 in these cells points out that type II pneumocyte differentiation is not well developed in the pale cells, and it is not clear that the neoplastic elements of SH closely recapitulate any of the normal epithelial cell types of the lung.

The staining of the papillary lining cells suggests that 2 different cell types may give rise to the papillary structures in SH. Some of the papillae were lined by cells cytologically similar to the pale cells of the solid areas. These papillary cells also stained in a similar fashion, with strong positivity for TTF-1, weaker staining for EMA, and generally negative or faint staining with CAM 5.2 and PE10. At the periphery of the tumors, the papillary lining cells were smaller, hobnailed, and closely resembled metaplastic type II pneumocytes. These cells demonstrated a close antigenic similarity to type II pneumocytes, with more consistent staining with CAM 5.2 and PE10, in addition to EMA and TTF-1. Thus, it appears that the smaller, hobnailed papillary lining cells at the periphery of the tumors indeed represent entrapped metaplastic type II pneumocytes (similar to those found in intrapulmonary mesenchymal tumors such as solitary fibrous tumor). Cuboidal papillary cells resembling the pale cells and staining similarly likely represent a papillary configuration of the neoplastic component. This suggestion may help explain some of the discrepancies in interpretation of the nature of the papillary lining cells. Further studies using microdissection and DNA analysis are needed to determine the validity of this hypothesis.

In summary, we believe that the uniform and strong positivity for TTF-1 in both pale cells and cuboidal cells supports the notion that this neoplasm shows evidence of type II pneumocyte differentiation.

Table 1. Type, Source, and Working Dilution of Antibodies and
Pretreatment of Slides Used in This Study

                                          Pre-
                Antibody     Dilution   treatment   Source

CD31           Monoclonal     1:1000    Citrate     Dako Corporation,
                 mouse Ig                 buffer      Carpinteria,
                 [G.sub.1]                            Calif
Epithelial     Monoclonal     1:1000    Citrate     Dako
  membrane       mouse Ig                 buffer
  antigen        [G.sub.2]
                 a, K
CAM 5.2        Monoclonal     1:1000    Citrate     Becton Dickinson,
                 murine Ig                buffer      San Jose, Calif
                 [G.sub.2]
Thyroid        Monoclonal     1:100     Citrate     Dako
  trans-         mouse Ig                 buffer
  cription       [G.sub.1]
  factor-1
PE10           Monoclonal     1:1000    Citrate     Dako
                 mouse Ig                 buffer
                 [G.sub.2]
                 b, K
Chromogranin   Monoclonal     1:5000    Citrate     Boehringer
                 mouse,                   buffer      Mannheim,
                 IgG, K                               Indianapolis,
                                                      Ind
Table 2. Summary of Immunohistochemical Staining of Pale Cells,
Cuboidal Cells Lining Papillary Structures That Resemble Pale
Cells, and Hobnailed Cuboidal Cells Lining Peripheral Papillary
Structures *

                                   Pale Cell-like      Hobnailed
                   Pale Cells      Cuboidal Cells    Cuboidal Cells

TTF-1            Positive,         Positive,         Positive,
                     nuclear           nuclear           nuclear
                     diffuse           diffuse           diffuse
                     strong (9)        strong (9)        strong (9)
EMA              Positive,         Positive,         Positive,
                     membranous        membranous        membranous
                     (9)               (9)               (9)
                   Diffuse           Diffuse           Diffuse
                     strong (1),       strong (2),       strong (9)
                     diffuse           diffuse
                     weak (2),         weak (2),
                     focal             focal
                     strong (3),       strong (1),
                     focal weak        focal weak
                     (3)               (4)
PE10             Negative (3)      Negative (1)      Positive (8)
  ([dagger])     Positive (5)      Positive (7)        Diffuse
                   Focal strong      Focal strong        strong
                     membranous        membranous        membranous
                     (1), focal        (2), focal        (8)
                     weak              weak
                     membranous        membranous
                     (1), focal        (5)
                     weak
                     cytoplasmic
                     (3)
CAM 5.2          Negative (2)      Negative (2)      Positive (9)
                 Positive (7)      Positive (7)        Diffuse
                   Focal weak        Diffuse weak        strong
                     membranous        membranous        membranous
                     (4), focal        (6), focal        (9)
                     weak              strong
                     cytoplasmic       membranous
                     (3)               (1)
CD31             Negative (8)      Negative (8)      Negative (8)
  ([dagger])
Chromogranin A   Negative (8)      Negative (8)      Negative (8)
  ([dagger])

* Values in parentheses indicate number of cases. TTF-1 indicates
thyroid transcription factor-1; EMA, epithelial membrane antigen.

([dagger]) PE10, CD31, and chromogranin A were not performed in 1 case.

References

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Accepted for publication June 1, 2001.

From the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY (Drs IIlei and Klimstra); and the Dipartimento di Patologia, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy (Dr Rosai).

Reprints: David S. Klimstra, MD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.