Microphthalmia

Context.-Osteoclast-like giant cells (GCs) in giant cell tumors (GCTs) are thought to derive from a monocyte-macrophage lineage. Microphthalmia transcription factor (MITF) is necessary for osteoclast gene expression and tartrate-resistant acid phosphatase (TRAP) activation; c-Kit plays a role in regulation of MITF.

Objective.-To gain insight into the differentiation of GCTs of bone (GCTBs) and GCTs tendon sheath (GCTTSs) by investigating immunohistochemical staining for c-Kit, MITF, TRAP, and HAM-56 in the GCs and stroma.

Design.-Immunoreactivity for CD117 (c-Kit), MITF, TRAP, and HAM-56 was studied in 35 GCTBs, 15 GCTTSs, and 5 foreign-body GC controls.

Results.-Across tumors, MITF and TRAP but not c-Kit were generally expressed in GCs; TRAP was variably expressed in stromal cells. The MITF was expressed more consistently in stromal cells of GCTTSs than GCTBs (P

Conclusions.-Results suggest that MITF and TRAP are expressed during osteoclast differentiation and that a proportion of mononuclear cells in GCTs express the macrophage marker HAM-56. Both GCTBs and GCTTSs show similar patterns of immunohistochemical expression.

(Arch Pathol Lab Med. 2005;129:360-365)

Osteoclast-like giant cells (GCs) are postulated to derive from the monocyte-macrophage lineage,1-6 and the GCs are believed to differentiate from mononuclear stromal cells.1 Molecules that are necessary for osteoclastogenesis are macrophage colony-stimulating factor4,5,7 and the receptor for activation of nuclear factor κB ligand.4,6,8 Macrophage colony-forming units, under the stimulation of macrophage colony-stimulating factor, proliferate to form monocyte-macrophage cells, which under continued stimulation by macrophage colony-stimulating factor and receptor for activation of nuclear factor κB ligand differentiate into osteoclast precursor mononuclear cells, which are tartrate-resistant acid phosphatase (TRAP) positive.4 These mononuclear cells, under further stimulation by macrophage colony-stimulating factor, receptor for activation of nuclear factor κB ligand, and interleukin 1, differentiate to multinucleated osteoclasts.4

Other important molecules in osteoclastogenesis are the microphthalmia transcription factor (MITF), a basic helix-loop-helix zipper factor, and the Ets family factor PU.1, expressed by hematopoietic cells, which act synergistically to activate the target gene TRAP.9-11 The MITF is expressed in osteoclast progenitor cells,2 and its expression during melanocyte differentiation is modified by c-Kit signaling.12 Thus, c-Kit activation of MITF may play a role in promotion of TRAP-positive mononuclear cell and multinucleated osteoclast formation. It has been shown that, although osteoclast precursors originate from mononuclear cells in both c-Kit-dependent and c-Kit-independent modes,8,13 nonetheless, 90% of the precursors required cKit signaling for differentiation.8

Although giant cell tumors of bone (GCTBs) and giant cell tumors of tendon sheath (GCTTSs) are recognized as separate entities, both contain cells that appear to be multinucleated osteoclast-like cells and mononuclear cells. It has been postulated that giant cell tumors (GCTs) consist of 3 basic cell types, namely, osteoclast-like GCs, mononuclear cells, and a group of as yet uncharacterized tumorspecific cells.14 We studied immunohistochemical expression of CD117 (c-Kit), MITF, TRAP, and HAM-56 in GCTBs and GCTTSs to further define the sequence of differentiation in these lesions.

MATERIALS AND METHODS

Case Selection and Review of Pathologic Findings

All cases were selected from the archives of the Department of Pathology, Pennsylvania Hospital, and cases that were initially diagnosed as GCTBs and GCTTSs from 1999 to 2003 were reviewed in accordance with the institutional review board-approved protocol 801376. The selected cases included 35 GCTBs, 15 GCTTSs (14 localized, 1 diffuse), and 5 foreign-body GC reactions. Hematoxylin-eosin-stained slides were reviewed for histologie evidence of fibrosis, osteoid formation, and vascular invasion. Giemsa stain was performed on all cases to identify and control for mast cells, which express c-Kit.15 If mast cells were present, these foci were not evaluated when determining c-Kit immunoreactivity.

Immunohistochemical Staining

Formalin-fixed, paraffin-embedded tissue sections were cut at 3 µM, heated at 58°C, deparaffinized in xylene, and hydrated in a graded series of alcohols. Antigen retrieval was performed by microwaving in citrate (pH 6.0 buffer) or directly on the Ventana Discovery Immunostainer (Table 1). lmmunohistochemical analysis was performed on nondecalcified blocks using an indirect avidin-biotin complex system and diaminobenzidine as the chromogen. The slides were counterstained with hematoxylin and rehydrated using xylene. In the case of c-Kit, appropriate positive controls, including gastrointestinal stromal tumors, were used. Furthermore, to determine a working concentration, a dilution gradient (1:25, 1:50, 1:100, and 1:200) was performed using the CD117 (c-Kit) antibody. Appropriate staining of control cases was achieved at 1:200; this concentration was used in the study.

Statistical Analysis

Immunohistochemical staining was evaluated independently by 2 pathologists. Staining was evaluated in GCs and stromal cells for both intensity of staining and percentage of cells stained. Intensity of expression was graded as strong (3 + ), moderate (2 + ), weak (1 + ), or absent (O), and percentage of expression was categorized as less than 10%, 10% to 30%, more than 30% to 60%, more than 60% to 90%, and more than 90%.

For each antibody, the percentage of staining and staining intensity categories among tumor groups were compared nonparametrically using the Kruskal-Wallis test. For antibodies that demonstrated a significant difference in rank values, the percentage of staining and staining intensity categories were dichotomized according to the median value (more than median value vs median value or less) in the overall sample. Correlations between antibody percentage of staining and staining intensity categories were determined using Spearman correlation coefficients.

RESULTS

Patient Population

The 35 GCTBs were from 29 patients, with 6 being recurrences. Of the 35 GCTBs studied, 13 (plus the 6 recurrences) were from female patients, who ranged in age from 15 to 47 years (average age, 24.5 years), and 16 were from male patients, who ranged in age from 15 to 47 years (average age, 32.2 years). The 15 GCTTSs were from 14 patients, with 1 recurrence of a localized GCTTS, and included 10 female patients, who ranged in age from 14 to 76 years (average age, 48.2 years), and 4 male patients, who ranged in age from 19 to 58 years (average age, 36.5 years).

Of the recurrent GCTBs, 5 were in female patients (P = .03). The recurrent GCTTS was in a female patient. No tumors metastasized.

The sites of GCTBs were as follows: tibia, 11; femur, 9; humerus, 3; hand, 2; wrist, 1; sacrum, 1; fibula, 1; and pelvis, 1. The sites of localized GCTTSs were as follows: hand, 5; knee, 4; ankle, 3; and foot, 1; the diffuse GCTTSs were from the hip.

Pathologic Findings

The GCTBs demonstrated variable populations of mononuclear cells and multinucleated osteoclast-like GCs. Significant fibrosis (>40% of tumor) was present in 5 cases (14%); osteoid with benign-appearing osteoblastic rimming was present in 8 cases (22%). Vascular invasion was identified in 1 case. Foci of mast cells were identified by Giemsa stain in 15 cases (43%). The GCTTSs were characterized by a mixed population of mononuclear cells and multinucleated GCs, with varying amounts of xanthomatous macrophages, fibrosis, and hemosiderin deposition. Mast cells were identified in 8 cases (53%). All foreignbody GC controls contained varying numbers of GCs, histiocytes, and mast cells.

Immunohistochemical Analysis

In both GCTBs and GCTTSs, HAM-56 staining was present in mononuclear cells but not in GCs (Figures 1 and 2); positive staining for HAM-56 was present in the multinucleated cells of all foreign-body GC controls.

Generally, MITF was expressed in both mononuclear cells and GCs of GCTBs and GCTTSs, as well as in multinucleated foreign-body GCs. Staining was nuclear (Figures 1 and 2). The MITF strongly stained the nuclei of all xanthoma cells in GCTTSs. The MITF was more often expressed in mononuclear cells of GCTTSs than GCTBs (P

Membranous staining with c-Kit was identified as an antibody concentration of 1:25 and 1:50 in stromal cells but not GCs in some GCTBs and GCTTSs. However, staining was lost at dilutions of 1:100 and 1:200. The recommended dilution is 1:200, which appropriately stained the gastrointestinal stromal tumor controls. Results at 1:200 were used in the statistical analysis.

TRAP was generally expressed in GCs of both GCTBs and GCTTSs and GC reaction controls. TRAP was variably expressed in mononuclear cells of both tumor types; staining was cytoplasmic (Figures 1 and 2). The GCTBs showed more TRAP GC staining (P = .04, Table 3) than GCTTSs.

HAM-56 staining in mononuclear cells correlated with MITF expression by mononuclear cells (r2 = 0.6, P

COMMENT

Both GCTBs and GCTTSs express antigens of the monocyte-macrophage (HAM-56) and osteoclastic GC (MITF and TRAP) lineage, as well as c-Kit in patterns of expression, which help to elucidate the sequential differentiation of these tumors. The HAM-56 staining in mononuclear cells in GCTBs and GCTTSs supports a monocyte-macrophage origin, since HAM-56 is known to stain macrophages,16 although not exclusively. We observed that HAM-56 failed to stain the GCs of any tumor but did stain the multinuclear GC controls, suggesting a difference in antigen expression by GC tumors. Thus, as mononuclear cells differentiate into multinucleated GCs, HAM-56 staining is lost.

In our study, c-Kit was not expressed in mononuclear cells or GCs of either GCTBs or GCTTSs at an appropriate dilution of 1:200. c-Kit is present in pluripotent hematopoietic stem cells'7; however, studies have shown that its expression is low.18 A previous study by Gattei et al,19 using indirect immunofluorescence, found detectable c-Kit expression by bone marrow-derived human preosteoclastic mononuclear cells and by multinucleated osteoclasts of bone and of GCTBs. In addition, an in vitro study by Yamazaki et al8 demonstrated that osteoclast differentiation from precursor cells can occur in both c-Kit-dependent and independent modes and that most cells required cKit. A further study by Haydon et al14 investigated c-Kit expression by immunohistochemical analysis in 10 paraffin-embedded archival GCTB samples. They found that all 10 tumors stained strongly with c-Kit. Sequence analysis of 2 of the 10 samples for activating mutations failed to reveal alterations in the c-kit gene. Cell lines were generated from 4 of these tumors using fresh tissue. When cultured cells were exposed to ST1571, growth was inhibited at 10 µM and cell death occurred at 20 µM. Fibroblast controls demonstrated no growth inhibition or cell death. These investigations suggest that c-Kit is present during osteoclast differentiation.

c-Kit expression is not equivalent to functional activation.2" Furthermore, we and others have shown that the ability to stain for c-Kit may be concentration dependent.15.20 In a study of desmoid tumors, c-Kit positivity was found to be present at low dilutions and lost with higher dilutions.1520 In this study, a dilution gradient for c-Kit of 1:25, 1:50, 1:100, and 1:200 was performed, and results were reported with the 1:200 dilution, which is the manufacturer's suggested dilution for c-Kit (Dako Corporation, Carpinteria, Calif). Although some cells stained at dilutions of 1:25 and 1:50, nonetheless, staining for cKit was lost at the 1:100 and 1:200 dilutions. However, based on the studies of Gattei et al19 and Yamazaki et al,8 the question of c-Kit relevance in osteoclastogenesis still remains. Additional assays, such as c-kit gene expression analysis, immunoblotting, and a more extensive mutation screening, would be useful in future studies to investigate the presence of a possible c-Kit alteration in GCTs.

The MITF is expressed in melanocytes,21 and others have shown that activation of MITF is c-Kit dependent.12,22 Osteoclast promotion from mononuclear cells is also at least partially c-Kit dependent.8 The MITF is known to act with PU. 1 to activate TRAP during osteoclastogenesis.9,10 seethala et al21 studied MITF expression in 72 GC lesions. They found that MITF was widely expressed in GCs and adjacent mononuclear cells in all GC lesions but was not expressed in mononuclear cells in lesions that did not contain multinucleated GCs. They concluded that MITF might play a role in the differentiation of mononuclear cells to form GCs in GCTs. In our study, MITF was generally expressed in both mononuclear cells and GCs of GCTBs and GCTTSs, as well as in multinucleated foreign-body GCs.

TRAP expression has been shown in vitro to be acquired during osteoclastogenesis.4 In our study, TRAP was expressed by both GCs and mononuclear cells, suggesting that the antigen is acquired in vivo during the monocytic osteoclast precursor phase of differentiation of GCT. TRAP expression in the GCTs of our study persisted in the fully differentiated multinuclear GCs. Suda et al,4 who reviewed previous studies of osteoclast differentiation and TRAP expression, found similar conclusions. Similarly, MITF, which is important for osteoclastic differentiation and activation of TRAP,9,10 was expressed in both mononuclear cells and GCs of both GCTBs and GCTTSs.

In summary, the results of this study suggest that MITF and TRAP are expressed during osteoclast differentiation and that a proportion of mononuclear cells in GCTs expresses the macrophage marker HAM-56. Based on the observations in our study and the model previously published by Suda et al,4 we propose a simplified schematic model of osteoclastic differentiation in GCTTSs (Figure 3). We propose that these lesions differentiate from macrophage-monocyte precursors and that MITF activation drives the genetic expression of TRAP in osteoclastic mononuclear precursors and multinucleated GCs. Any role of c-Kit remains unclear. Similar patterns of immunohistochemical expression in GCTBs and GCTTSs suggest a similar differentiation sequence.

We thank Anna Marie McClain for her meticulous assistance with the manuscript.

References

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23. Seethala RR, Goldblum ]R, Lehman M, et al. Immunohistochemical evaluation of MITF expression in giant cell lesions. Mod Pathol. 2003;16:20A.

Rolando Y. Ramos, MD; Helen M. Haupt, MD; Peter A. Kanetsky, PhD; Rakesh Donthineni-Rao, MD; Carmen Arenas-Elliott, MS; Richard D. Lackman, MD; Anne-Marie Martin, PhD

Accepted for publication October 29, 2004.

From the Departments of Pathology (Drs Ramos, Haupt, Arenas-Elliott, and Martin), and Orthopedic Surgery (Dr Lackman), Pennsylvania Hospital, Philadelphia; the Department of Biostatistics and Epidemiology and Center for Clinical Epidemiology and Biostatistics (Dr Kanetsky), Department of Orthopedic Surgery (Dr Lackman), and Department of Medicine (Dr Martin), University of Pennsylvania, Philadelphia; and Department of Orthopedic Surgery, University of California, Davis (Dr Donthineni-Rao).

The authors have no relevant financial interest in the products or companies described in this article.

Corresponding author: Helen M. Haupt, MD, Pennsylvania Hospital, 800 Spruce St, Philadelphia, PA 19107 (e-mail: haupt@pahosp.com).

Reprints not available from the authors.

Copyright College of American Pathologists Mar 2005
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