Abstract
Background: Macrophage colony stimulating factor (M-CSF) binds to colony-stimulating factor-1 receptor (CSF-1R) and thereby stimulates the proliferation, differentiation and behaviour of monocytes, macrophages and their bone marrow progenitors. Previous studies have suggested that high expression of these markers is correlated with poor prognosis. Materials and Methods: M-CSF, CSF-1R and CD68 protein expression was examined by immunohistochemistry in paraffin embedded sections of soft tissue tumor specimens from 46 patients. The proportion of positive cells and the expression intensity of M-CSF, CSF-1R and CD68 in both the tumor cell areas and the adjacent stromal areas were correlated to the histological grade. Results: In the high grade tumors M-CSF and CSF-1R were more highly expressed than in the low grade tumors. This was seen in both the tumor cell areas and the adjacent stromal areas. No differences in CD68 expression between the high and low grade tumors were found either in the tumor cell areas or the stromal areas. Conclusion: The expression of M-CSF and CSF-1R in tumor cell areas and adjacent stromal areas correlate with the histological grade of soft tissue tumors.
Soft tissue tumors are relatively rare tumors representing approximately 1% of human carcinomas. They occur at different body sites and vary greatly in their aggressive abilities. Although several prognostic factors (i.e. tumor size, site, depth and histological type) are associated with the metastatic potential of these tumors, tumor grade remains as the single most reliable prognostic indicator (1). About 50% of patients with soft tissue tumors die because of their disease (2). Neoplastic cells produce a wide spectrum of cytokines and chemokines that attract leukocytes. At a site of tissue injury, monocytes migrate to the site of injury guided by chemotactic factors. Once activated, macrophages are the main source of growth factors and cytokines (3). Tumor-associated macrophages (TAMs) have powerful effects in the neoplastic process because they can adopt tropic roles and are educated by the tumor microenvironment to facilitate angiogenesis, matrix breakdown and tumor cell motility (4). Altogether, this gives malignant neoplasms the capacity to invade normal tissues and metastasize to and grow in distant body parts (4, 5). Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of monocytes to macrophages. In inflammation M-CSF induces macrophages to secrete cytokines and proteases, thereby enhancing the macrophages' ability to combat microbial infections (6). Macrophage colony stimulating factor 1 receptor (CSF-1R) is one of the growth factor receptors which regulate the proliferation and differentiation of the monocyte lineage (7, 8). In the inflammatory stromal microenvironment of neoplastic tissues, the M-CSF and CSF-1R are important bioactive proteins in a diversity of human carcinomas (7, 9, 10). M-CSF has mainly been studied in breast carcinomas where it is commonly expressed. Increased expression of M-CSF and CSF-1R is correlated with poor prognosis in breast, ovarian and prostate cancer (11-14). Experimental studies in sarcoma cell lines have revealed a paracrine loop in the regulation between tumor cells secreting M-CSF and the activity of macrophages (15). Moreover, mononuclear osteoclast precursors have been found in the macrophage population of cells isolated from Ewing's sarcoma and these cells were able to differentiate into osteoclasts capable of bone resorption in the presence of M-CSF (16). In a previous study we showed that the high expression of M-CSF and CSF 1R in primary prostate cancer was associated with metastatic potential (17). In this study the relationship between M-CSF, CSF-1R and CD68 (macrophage marker) expression and histological grade in soft tissue tumors, was explored.
Clinicopathological characteristics of 46 adult patients with soft tissue tumors.
Materials and Methods
Patients and samples. Tumor tissue from 39 patients with previously untreated soft tissue sarcomas and 7 patients diagnosed with gastrointestinal stromal tumor (GIST) were included. All were diagnosed at the Prince of Wales Hospital, Sydney, Australia. This study was approved by the Research Ethics Committee of the South Eastern Sydney Area Health Service (REES, Ref.:00/253).The patients'clinical and pathological information is shown in Table I. The primary histological diagnostic confirmation was by ‘Trucut’ or open excision biopsy, according to the classification of Einzinger and Weiss (18). Except for the GIST, all the tumors were graded as Grade 1, 2 or 3, according to a scoring system based upon the scheme from the French Federation of Cancer Centers Sarcoma Group (FNCLCC) (19-22), and into high grade (FNCLCC grade 2 and 3) or low grade (FNCLCC grade 1) according to the TNM classification (23). The FNCLCC system incorporates standard histopathological parameters including degree of differentiation, mitotic count and microscopically assessed necrosis. The tumor depth was classified as superficial or deep to the fascia (23). The GIST specimens were classified according to WHO criteria (24). This system incorporates standard histological parameters as the degree of differentiation, which includes low, moderate and high grade. The system also includes a mitotic index and mutation analysis of C-KIT (CD117), which is a cytokine receptor, expressed on the surface of hematopoietic stem cells as well as other cell types. Mutation in this gene is associated with GIST (25). In the statistical analyses, the TNM classification (low grade vs. high grade) was used. All the specimens were scored separately by to experienced pathologists (ER and SWS) who were blinded to each other and to the clinical information.
Immunohistochemical expression of M-CSF, CSF-1R and CD68 in tumor cell areas.
Immunohistochemical expression of M-CSF, CSF-1R and CD68 in tumor adjacent stromal areas.
High-grade gastrointestinal stromal tumor (GIST). A, M-CSF strong staining in neoplastic tumor tissue and negative in fibrous and fat tissue (100*). B, Strong cytoplasmic staining of M-CSF in tumor cells (400). C, CSF-1R expression in neoplastic tumor areas adjacent to negative stromal areas with inflammatory cells (100). D, CSF-1R strongly expressed in tumor cells (400). *Magnification.
Immunohistochemistry (IHC). IHC was performed on phosphate-buffered saline (PBS)-formalin fixed, and paraffin-embedded tissue sections (4 μ) using standard protocol. The sections were deparaffinized with xylene and rehydrated through a graded series. The applied antibodies had been in-house validated by the manufacturer for IHC analysis on paraffin-embedded material. For antigen retrieval for M-CSF and CSF-1R (both diluted 1:50 in PBS), the sections were placed in a microwave oven with Tris/EDTA buffer, pH 9.0 for intervals of 2×10 min at 450 W. For CD68 (diluted 1:100 in PBS), citrate buffer, pH 7.0, was used for intervals of 2×10 min at 450 W. The slides were transferred to a Ventana Benchmark, XT automated slide stainer (Ventana Medical System, France). The tissue sections were incubated with primary antibodies recognizing M-CSF, CSF-1R (both rabbit polyclonal antibodies, Santa Cruz Biotechnology, CA, USA), and the macrophage marker CD68 (a mouse monoclonal antibody, Ventana). Biotinylated goat anti-mouse IgG and mouse anti-rabbit IgM, both 200 μg/ml, were used as the secondary antibodies. The Peroxidase Block from a DAKO EnVision+ System, peroxidase (DAKO Envision+ System, HRP) was used for endogenous peroxidase blocking. This system is based on an HRP labelled polymer which is conjugated with secondary antibodies. In summary, to quench endogenous peroxidase activity the specimens were incubated with DAKO Peroxidase Block for 5-10 minutes (room temperature). Then the specimens were incubated with primary mouse- or rabbit antibody, followed by incubation with labelled polymer for 30 minutes. Finally the specimens were incubated by diaminobenzidine (DAB)+ substrate-chromogen for 5-10 minutes, which resulted in brown-coloured precipitate at the antigen site. All the slides were then counterstained with hematoxylin to visualize the nuclei. As negative staining controls, the primary antibodies were replaced with the primary antibody diluents. Appropriate positive and negative controls were included in each antibody run according to the manufacturer's recommendations. The stromal areas were defined as the stromal tissue adjacent to the tumor areas. For stromal cell characterisation the slides were stained by Masson Trichrome (collagen fibres) (26), Giemsa (granulocytes), CD34 (vessels), CD20 (B-lymphocytes), CD3, CD8 and CD4 (T-lymphocytes), CD68 (macrophages), CD56 (NK-cells) and CD1a (dendritic cells) (all antibodies were from Ventana). The staining intensity of M-CSF, CSF-1R and CD68 was scored as 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong staining).
Statistical analysis. All the data are expressed as means±SD Differences in expression between low grade and high grade tumors were determined by Mann-Witney U-test, Kruskall-Wallis test and one way ANOVA. Differences in expression of M-CSF, CSF-1R and CD68 between tumor cell areas and tumor stromal areas were tested by Wilcoxon signed rank test. Individual groups were compared using the Student's t-test. The interobserver Kappa-value for M-CSF (tumor cell areas and stromal areas) were 0.62 and 0.44, for CSF-1R 0.60 and 0.83, and for CD68 0.82 and 0.81. A two-sided p-value <0.05 was considered significant. The SPSS/Win 14.0 statistical program (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis.
Results
Pathological and demographic data. The mean age of the patients was 55.7 years (range 16-90 years). Myxofibrosarcoma/pleomorphic sarcoma (MFH, myxoid-type/MFH, pleomorphic type), leiomyosarcoma and gastrointestinal stromal tumors (GIST) were the most common tumor types (Table 1). According to the TNF classification, ten tumors were low grade and 36 were high grade. The mean maximum tumor diameter was 7.5 cm (range 0.8-27 cm).
Expression of M-CSF, CSF-1R and CD68. Figures 1 and 2 demonstrate the IHC expression of M-CSF, CSF-1R and CD68 in tumor cell areas and adjacent stromal areas of high grade and low grade malignancies. In all the high grade tumors together, M-CSF was significantly higher expressed in the tumor cell areas (p=0.028) and was borderline correlated in the adjacent stromal areas (p=0.09), as compared to the low grade tumors. The CSF-1R expression in the high grade tumors was significantly higher in both the tumor cell areas (p=0.005) and the stromal areas (p=0.002) compared to the low grade tumors. The M-CSF and CSF-1R staining was predominantly cytoplasmic (Figure 3). In the tumor areas the M-CSF and CSF-1R were expressed by the tumor cells. The expression of M-CSF and CSF-1R in the adjacent stromal areas was mainly detected in cells with morphological resemblance to macrophages, lymphocytes and fibroblasts. No differences in CD68 expression were found between the groups, either in the tumor cell or stromal areas.
The high-grade leiomyosarcomas showed significantly higher expression of M-CSF (p=0.011) and CSF-1R (p=0.023) in both tumor cell and stromal areas, compared to the low-grade leiomyosarcomas. No differences were found for CD68. Moreover, the high-grade GISTs, had higher expression of CSF-1R compared to the low-grade GIST tumors (p=0.023), whereas for M-CSF and CD68, there was a non-significant trend (p=0.098 and p=0.083) towards higher expression in the high-grade tumors. For the other histological entities, no significant correlations were found.
Discussion
In this study, higher expressions of M-CSF and CSF-1R in both the tumor cell areas and the adjacent stromal areas were found in the high-grade tumors compared to the low-grade tumors. To our knowledge, this was the first study reporting the expression of M-CSF, CSF-1R and CD68 of soft tissue tumors of different histological grades and distinguishing between tumor cell and stromal compartments.
The diagnosis of soft tissue tumors can be difficult. Russel et al. (27) were the first to recognize histological grade as the most important prognostic factor in these tumors. Others have shown that the histological grade predicted metastases (28-30) and in large series of patients histological grade was found to predict survival time (31, 32). The goal of a grading system is primarily to separate tumors of poor prognosis (high grade) from those of good prognosis (low grade) (33). The identification of molecular markers specific to early and late events in cancer progression will hopefully have implications for risk stratification, cancer detection, therapy and outcome. Cancer cells are dependent on the stroma for proliferation, progression and metastasis and the intercellular communication between cancer cells and their surrounding stroma involves a number of different factors and receptors.
The role of M-CSF and CSF-1R in tumor progression has been extensively studied (12-14). In breast cancer, increased infiltration of macrophages correlates with increased tumor growth and metastasis. M-CSF and CSF-1R are stimulators of many cell types involved in immunological responses and act especially as regulator molecules of macrophage proliferation, differentiation and survival (35-37). Lin et al. (38) crossed transgenic mice susceptible to mammary cancer with mice containing a recessive null mutation in the M-CSF gene before inducing transgenic M-CSF expression in the mammary epithelium. By this model they demonstrated that M-CSF expression did not affect the initiation of growth of primary tumors, but rather promoted tumor acceleration to late stages and the development of metastasis. Many carcinomas arise from sites of infection or chronic inflammation which activate macrophages and leucocytes promoting tumorgenesis, tumor angiogenesis and metastasis (4). Macrophages are the first line of defence against invading pathogens and are frequently found in the stromal areas surrounding carcinomas (39-41). In the present sample population higher expression of M-CSF, CSF-1R was shown in both the tumor cell areas and the tumor stromal areas, while for CD68 no significant correlations were found between the histological grades. This may indicate a complex pathway of macrophage regulation, but the lack of correlations could also be explained by the limited number of patients.
In conclusion, M-CSF and CSF-1R expression is correlated to histopathological grade in soft tissue tumors. This finding may be of importance in predicting malignant potential in these tumors
Acknowledgements
This work was supported by The Norwegian Cancer Society and the Research Fund of the University of New South Wales, Australia. We thank Dr. Peter Bulpitt, Dr. Claire Cooke-Yarborough and Tone Bjørnsen for their excellent assistance.
Footnotes
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Conflict of Interest
There are no conflicts of interest concerning this paper.
- Received April 21, 2009.
- Revision received July 6, 2009.
- Accepted July 15, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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