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Clinical Cancer Research Vol. 10, 5804-5807, September 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Expression of Transforming Growth Factor ß Isoforms and Their Receptors in Malignant Fibrous Histiocytoma of Soft Tissues

Tetsuji Yamamoto1, Toshihiro Akisue1, Takashi Marui1, Ikuo Fujita2, Keiji Matsumoto2, Toshiaki Hitora1, Teruya Kawamoto1, Keiko Nagira1, Tetsuya Nakatani1 and Masahiro Kurosaka1

1 Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe; and 2 Department of Orthopaedic Surgery, Hyogo Medical Center for Adults, Akashi, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Transforming growth factor ß (TGF-ß) is a multifunctional growth factor that variably affects proliferation, differentiation, and extracellular matrix formation. Little information is currently available on the TGF-ß expression in malignant fibrous histiocytoma (MFH). The aims of the present study were to investigate the expression of TGF-ß isoforms and their receptors in human MFH specimens.

Experimental Design: The expression of TGF isoforms, and TGF-ß receptors (TGF-ßR1 and -ßR2) were immunohistochemically evaluated in 43 paraffin-embedded MFH specimens. Furthermore, the correlation of the TGF-ß and receptor expression with tumor proliferative activity assessed by MIB-1 indices was analyzed.

Results: Positive immunoreactivity for TGF-ß1, -ß2, and -ß3 was identified in tumor cells of 42, 40, and 38 of the 43 MFHs, respectively. In each TGF-ß isoform immunostaining, the specimens were divided into two groups based on the number of positive tumor cells: those with low (<25%) and those with high (greater double equals25%) immunoreactivity. There were no statistically significant differences in the MIB-1 indices between the two groups. Positive immunoreactivity for TGF-ßR1 and -ßR2 was identified in tumor cells of 36 and 24 of the MFHs, respectively. The specimens were divided into two groups based on their receptor expression patterns: those with both TGF-ßR1- and -ßR2-positive immunoreactivity (n = 23), and those with both or either TGF-ßR1- and -ßR2-negative immunoreactivity (n = 20). The MIB-1 indices in the both-TGF-ßR1- and -ßR2-positive group were significantly higher than those in the other group (P = 0.0102). There was no significant difference in pulmonary metastasis ratios between the two groups.

Conclusions: These findings strongly suggest an association of the TGF-ß ligand/receptor system with a significantly higher MIB-1 index in human MFHs. Investigation of the TGF-ßR1 and -ßR2 coexpression might be useful in predicting tumor behavior of MFHs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor ß (TGF-ß) is a multifunctional cell regulatory peptide that variably affects proliferation, differentiation, tissue repair, and extracellular matrix formation. To date, at least three TGF-ß isoforms (TGF-ß1, -ß2, and -ß3) have been identified in mammalian tissues and are known to biologically interact with their two different receptors (TGF-ßR1 and TGF-ßR2; refs. 1 and 2 ). TGF-ß usually mediates cell growth suppression (3) but may paradoxically play an important role in promoting the development of several malignant diseases (4 , 5) . Several investigators have demonstrated the TGF-ß overexpression in cultured cancer cell lines and their localization in tumor tissues and have suggested that TGF-ß has a potent stimulatory effect on the growth of malignant epithelial neoplasms (6, 7, 8, 9, 10) . As for mesenchymal neoplasms, TGF-ß expression has been investigated in some osteocartilaginous-matrix-producing tumors, including osteosarcomas (11 , 12) and chondrosarcomas (13) . TGF-ß regulates bone and cartilage formation by inducing osteoblast and osteoclast proliferation and by inducing extracellular matrix formation in normal and neoplastic tissues (14 , 15) .

However, little information is currently available on the prevalence and distribution of TGF-ß isoforms and their receptors in soft tissue tumors of a fibrohistiocytic origin, including malignant fibrous histiocytoma (MFH). MFH is one of the most common soft tissue sarcomas in adult life and has an aggressive behavior and a high metastatic potential to other organs (16) . The aims of the present study were to investigate the endogenous expression of TGF-ß isoforms and their receptors in human MFHs by immunohistochemical techniques. Furthermore, we analyzed the correlation of TGF-ß expression in MFHs with proliferative activity assessed by MIB-1 immunohistochemical staining.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Specimens.
Tissue specimens from 43 soft tissue MFH cases were selected from the files of the pathology departments in the present authors’ institutions. All of the specimens were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. There were 21 female and 22 male patients. The patients ranged in age from 17 to 83 years (mean, 61 years). The tumors were located in the upper extremity (n = 10), the lower extremity (n = 28), and the trunk (n = 5). The specimens were obtained from biopsies or resections before chemotherapy. Four-micrometer serial sections were prepared for staining with hematoxylin and eosin, and for the immunohistochemical studies. After evaluating the specimens stained with hematoxylin and eosin, all of the cases were confirmed to conform to the diagnostic histologic criteria of MFH proposed by Enzinger and Weiss (16) .

The mean follow-up period was 63 months. Of the 43 patients with MFH, 19 patients developed pulmonary metastases postoperatively, and one patient was found to have pulmonary metastasis at the initial presentation.

Immunohistochemistry.
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections by the indirect immunoperoxidase method. Briefly, the sections were deparaffinized with xylene and were routinely dehydrated through a series of graded alcohols. Antigen unmasking in the sections was performed by autoclaving pretreatment for 15 minutes. After elimination of endogenous peroxidase activity with a 10-minute incubation in 3% H2O2, the sections were incubated at 4°C overnight with primary antibodies against TGF-ß1 (15 µg/mL, monoclonal, Genzyme-Techne, Minneapolis, MN), TGF-ß2 (6 µg/mL, polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), TGF-ß3 (6 µg/mL, polyclonal, Santa Cruz Biotechnology), MIB-1 (prediluted, monoclonal, Dako Japan, Kyoto, Japan), or at room temperature for 1 hour with primary antibodies against TGF-ßR1 (6 µg/mL, polyclonal, Santa Cruz Biotechnology) and TGF-ßR2 (1 µg/mL, polyclonal, Upstate Biotechnology, Lake Placid, NY). After washing with Tris-buffered saline (TBS), the sections were then incubated at room temperature for 40 minutes with goat antimouse immunoglobulins (antirabbit immunoglobulins for TGF-ßR1 and -ßR2) conjugated to peroxidase-labeled dextran polymer (Envision+, horseradish peroxidase, Dako Japan). 3,3'-diaminobenzidine tetrahydrochloride was used for color development, and the sections were counterstained with hematoxylin. Autoclaving pretreatment was not performed for the immunostaining of TGF-ßR1 and -ßR2. Negative controls were obtained by substituting the primary antibodies with nonimmune rabbit or mouse serum. Paraffin sections of human growing articular cartilage tissues were used as positive controls. In TGF-ß isoform immunostaining, an absorption test was performed to exclude nonspecific staining by the blocking of immunoreactivity by the use of their respective immunizing peptides.

Evaluation of Immunohistochemistry.
A semiquantitative system was used to evaluate the level of antigen expression; immunoreactivity was scored as negative (–), focal (1+, less than 25% of positive tumor cells), moderate (2+, 25–50% of positive tumor cells), or diffuse (3+, more than 50% of positive tumor cells). The percentage of MIB-1-positive cells (MIB-1 proliferative index) was determined by examining 20 high power fields in representative areas. In each case, at least 500 viable tumor cells were counted.

The statistical significance of the individual findings and their association indices were evaluated by Mann-Whitney U test or {chi}2 test. Probability (P) values less than 0.05 were considered to be significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical results of TGF-ß isoforms and their receptors are shown in Table 1Citation . Positive cytoplasmic immunoreactivity for TGF-ß1, -ß2, and -ß3 was identified in tumor cells of 42 (98%), 40 (93%), and 38 (88%) of the 43 MFH cases, respectively (Fig. 1)Citation . All of the MFHs analyzed showed positive immunoreactivity for one or more TGF-ß isoforms. All of the TGF-ß isoforms were expressed in 38 of the 43 MFH cases studied. Positive cytoplasmic immunoreactivity for TGF-ßR1 and -ßR2 was observed in tumor cells of 36 (84%) and 24 (56%) MFH cases, respectively (Fig. 2)Citation . Both TGF-ßR1 and -ßR2 were expressed in 23 cases (54%).


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  		Table 1 Expression of TGF-ß isoforms and their receptors in MFH

 


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  		Fig. 1. Tumor cells of a MFH show diffuse cytoplasmic immunoreactivity for TGF-ß1 (immunostain, x200).

 


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  		Fig. 2. Tumor cells of an MFH show diffuse cytoplasmic immunoreactivity for TGF-ßR1 (immunostain, x400).

 
Overall, in the MFH cases analyzed, the MIB-1 proliferative indices ranged from 2.5 to 74% (mean, 25.8%). In each TGF-ß1, -ß2, and -ß3 immunostaining, the specimens were divided into two groups based on the number of positive tumor cells; those with low (–, 1+) and those with high (2+, 3+) immunoreactivity. There were no significant differences in MIB-1 indices between the low and high immunoreactivity groups in any of the TGF-ß1, -ß2, or -ß3 immunostaining (Table 2)Citation .


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  		Table 2 Comparison of MIB-1 indices between low and high expression groups for TGF-ß isoforms

 
Because both TGF-ßR1 and -ßR2 are needed to mediate a growth-regulation signal, the specimens were divided into two groups based on their receptor expression patterns: those with both TGF-ßR1 and -ßR2-positive immunoreactivity (n = 23), and those with both or either TGF-ßR1 and/or -ßR2-negative immunoreactivity (n = 20). The MIB-1 indices in the both-TGF-ßR1 and -ßR2-positive group were significantly higher than those in the other group (Mann-Whitney U test, P = 0.0102; Table 3Citation ).


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  		Table 3 Correlation of MIB-1 indices with the TGF-ß receptor expression patterns in MFH

 
In the group in which there was both TGF-ßR1- and the TGF-ßR2-positive immunoreactivity, 9 patients developed pulmonary metastases; in the other group, 11 patients developed pulmonary metastases. There was no significant difference in the pulmonary metastasis ratios between the two groups ({chi}2 test).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGF-ß is a growth factor that differently influences cell proliferation according to the target cell type. Effects of TGF-ß on cell proliferation are inhibitory for epithelium-derived cells, and stimulatory for some mesenchyme-derived cells (3 , 11 , 17) . The increased expression of TGF-ß has been reported in various epithelial neoplasms, including colon (6) , breast (7) , pancreatic (8) , gastric (10) , and endometrial carcinomas (9) . Furthermore, this increased TGF-ß expression is related to tumor progression and decreased survival, which suggests cell growth via an autocrine/paracrine loop in these malignant tumors (7 , 11 , 18) .

There have been several reports on the TGF-ß expression in mesenchymal tumors. Kloen et al. (11) immunohistochemically investigated the TGF-ß expression in 25 osteosarcomas and revealed that all of the osteosarcomas studied showed positive immunoreactivity for one or more TGF-ß isoforms. They demonstrated that intense immunoreactivity for TGF-ß3 was related to disease progression. Franchi et al. (12) reported that all of the high-grade osteosarcomas that they analyzed expressed TGF-ß1, -ß2, and -ß3, and that high-grade osteosarcomas had a significantly higher expression of TGF-ß1 than did low-grade osteosarcomas. Yang et al. (19) also reported that one or more TGF-ß isoforms were identified in tumor cells of all of the 16 osteosarcomas studied, and that the expression of TGF-ß1 or -ß3 was associated with a higher rate of lung metastasis of the tumors. Ciernik et al. (5) found that tumor cells of most of the Kaposi’s sarcomas produced TGF-ß. Masi et al. (13) reported that each TGF-ß1, -ß2, and -ß3 was expressed in 70–80% of the chondrosarcomas analyzed, with a higher expression in the high-grade tumors than in the low-grade tumors. Most investigators showed positive correlations of TGF-ß expression with aggressiveness of the mesenchymal tumors and clinical prognosis.

In the present study, we have immunohistochemically shown that tumor cells of many MFHs have the ability to produce TGF-ß isoforms. All of the MFHs studied expressed one or more TGF-ß isoforms. The absorption tests in TGF-ß isoform immunostaining confirmed that the staining was specific. To our knowledge, there have been no reports describing the expression and distribution of TGF-ß isoforms in human MFHs. We compared tumor cell proliferative activity assessed by MIB-1 indices between the tumors with low and high TGF-ß expression, but in all TGF-ß isoform immunostaining, there were no significant differences in MIB-1 indices between the two groups. Because of the limited number of TGF-ß negative tumors, statistical analysis of MIB-1 indices between the TGF-ß-positive and -negative tumors was not performed.

TGF-ßR1 and -ßR2 were expressed in 84 and 56% of the MFHs studied, respectively. Both receptors were expressed in 53% of the tumors. Masi et al. (13) reported that most (95.8%) of the chondrosarcomas investigated demonstrated both TGF-ßR1 and -ßR2. Ciernik et al. (5) reported that, in Kaposi’s sarcomas, TGF-ßR2 was expressed in 83% of the tumors investigated but that none of the tumors expressed TGF-ßR1. It is known that both receptors are needed to mediate the effects of TGF-ß on regulation of cell proliferation (20) . We compared MIB-1 indices in MFHs with both TGF-ßR1- and -ßR2- positive immunoreactivity with those in the remaining MFHs. The MIB-1 indices in the both-receptors-positive MFH group were significantly higher than those in the other MFH group. This finding strongly suggests that the TGF-ß ligand-receptor system plays an important role in promoting cell proliferation of human MFH tissues via an autocrine/paracrine loop. However, there was no significant difference in pulmonary metastasis ratios between the two groups. Because the clinical outcome of MFH is influenced by various factors including histologic grade and chemosensitivity of each tumor, multifactorial analyses of a large number of MFHs are required.

Previous studies indicated that TGF-ß is an important modulator of angiogenesis, with promotion of neovascularization in malignant neoplasms (21) . Furthermore, other biological effects of TGF-ß include immunosuppression (22) and alteration in the extracellular matrix (23) . These factors could indirectly influence tumor progression of MFHs in addition to having direct effects on cell growth.

In conclusion, we demonstrated that the human MFHs expressed TGF-ß isoforms in vivo, with the highest frequency occurring in TGF-ß1 expression. We found that MIB-1 proliferative indices significantly correlated with TGF-ßR1 and -ßR2 coexpression. Investigation of TGF-ßR1 and -ßR2 coexpression might allow oncologists to predict tumor behavior of MFHs. These observations suggest that the TGF-ß ligand-receptor system might be an important autocrine and/or paracrine cell growth regulator in human MFHs.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Tetsuji Yamamoto, Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5985; Fax: 81-78-351-6944; E-mail: yamatetu{at}med.kobe-u.ac.jp

Received 5/14/03; revised 3/31/04; accepted 5/18/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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