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Keywords:

  • chordoma;
  • hepatocyte growth factor;
  • c-MET;
  • epithelial differentiation;
  • proteinase;
  • invasion;
  • proliferation;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND.

Hepatocyte growth factor (HGF) is a multipotent cytokine that is mediated by its receptor, c-MET. HGF/c-MET contributes to tumor progression in many human malignancies; however, HGF/c-MET is inversely correlated with aggressive biologic behavior in other cancers. Conversely, to the authors' knowledge, little is known regarding the significance of HGF/c-MET expression in skull base chordoma.

METHODS.

Using immunohistochemical techniques, the authors investigated HGF/c-MET expression in 46 primary and 25 recurrent lesions, and compared it with the expression of proteinases and cell differentiation markers, proliferative ability, and other clinicopathologic parameters.

RESULTS.

c-MET was found to be expressed in 70.0% of primary and 88.0% of recurrent lesions. HGF expression was scarcely detected. Higher c-MET expression was found to be correlated with younger patient age. Lesions with a higher expression of low molecular weight cytokeratin (CAM5.2) demonstrated significantly higher c-MET scores in both primary and recurrent lesions compared with those with lower CAM5.2 expression. In recurrent lesions, higher c-MET expression was found to be associated with the scores of matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of matrix metalloproteinase-1, and urokinase plasminogen activator (uPA); however, only uPA was found to be correlated with higher c-MET expression in primary lesions. c-MET expression did not appear to be correlated with MIB-1 labeling index. Patients with higher c-MET expression were found to have longer survival.

CONCLUSIONS.

In the current study, c-MET expression was a common event, and was found to be correlated with CAM5.2 expression, younger patient age, and a favorable prognosis in patients with skull base chordoma. However, HGF/c-MET paracrine signaling also may contribute to its invasive ability, especially in recurrent lesions. Cancer 2008. © 2007 American Cancer Society.

Hepatocyte growth factor (HGF), which is produced by mesenchymal cells,1–6 was first identified as a major mediator of liver regeneration.7 It exerts diverse biologic effects on epithelial cells, including mitogenesis,8–11 morphogenesis,12, 13 and cell motility.5, 14 Conversely, the receptor for HGF, c-MET, is mainly expressed in epithelial cells.11, 15–18 Therefore, HGF and c-MET have been considered as a model for a paracrine signaling system in mesenchymal-epithelial interaction.19

c-MET, a transmembrane tyrosine kinase receptor, is encoded by the c-met proto-oncogene, which was originally identified as a transforming gene activated by a rearrangement in a human osteosarcoma cell line treated with a chemical carcinogen.20, 21 In addition, cotransfection of NIH-3T3 cells with met and HGF resulted in tumorigenesis.22 These findings suggest that HGF/c-MET is involved in the pathogenesis of neoplasms. In agreement with this hypothesis, the paracrine or autocrine HGF/c-MET signaling system has been reported to contribute to tumorigenesis and disease progression or to correlate with invasiveness and poor prognosis in many human carcinomas23–26 and sarcomas.27–30 However, in several human malignancies, HGF/c-MET is believed to play a role in the early stages of neoplastic promotion because c-MET expression not only decreases in poorly differentiated tumors31–34 but also is correlated with favorable prognosis.32–35 These findings suggest diverse roles of the HGF/c-MET signaling system in various types of tumors.

Chordoma, which is assumed to originate from notochordal remnants, is a unique bone tumor exhibiting both epithelial and mesenchymal differentiation. It grows slowly but demonstrates an invasive growth pattern, frequently recurring after surgical treatment. Frequent c-MET expression was previously reported in a small series of skull base and nonskull base chordomas.36, 37 However, the role of HGF/c-MET signaling remains controversial in skull base chordoma. The objective of the current study was to investigate the expression of both HGF and c-MET and to clarify their clinicopathologic significance, placing special emphasis on the correlation between HGF/c-MET signaling and cell differentiation, proliferative ability, invasive ability, and prognosis in skull base chordoma.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Tumors

A total of 71 skull base chordomas (46 primary and 25 autologous recurrent lesions; 29 male and 17 female patients) were used in this study. A total of 69 lesions (from 44 patients) were obtained from the Department of Pathology at Nordstadt Medical Center, Clinics of Hanover, Germany, and 2 additional lesions (from 2 patients) were provided by the Department of Pathology at Otto-von-Guericke University, Magdeburg, Germany. The age of the patients at the time of initial surgery ranged from 11 to 72 years (median age, 43 years). Histologic sections obtained at biopsy or surgically resected specimens were routinely stained with hematoxylin and eosin for diagnostic purposes (Fig. 1A). The following immunohistochemical studies were performed using sections from 10% formalin-fixed, paraffin-embedded tissues, highlighting the representative areas of the tumor.

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Figure 1. (A) Syncytial arrangements of tumor cells in the form of cords (H & E) and expression of (B) hepatocyte growth factor (score 1), (C) c-MET (score 3), and (D) CAM5.2 (score 1) in skull base chordomas.

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Light Microscopic Study

Nuclear pleomorphism, intratumoral necrosis, and intratumoral bleeding were evaluated as positive or negative. As previously reported, mitosis38–43 and apoptosis41–43 were scarce. Therefore, we also assessed them as positive or negative instead of calculating mitotic or apoptotic indices.

Immunohistochemical Study

The lesions were investigated immunohistochemically for the expression of HGF (polyclonal, dilution 1:25; Immuno-Biological Laboratories, Hamburg, Germany), c-MET (polyclonal, dilution 1:500; Santa Cruz Biotechnology, Heidelberg, Germany), pancytokeratin (AE1/AE3, dilution 1:200; BioGenex, San Ramon, Calif), low molecular weight cytokeratin (CAM5.2, dilution 1:5; Becton Dickinson, San Jose, Calif), vimentin (Vim3B4, dilution 1:400; Dako Corporation, Carpinteria, Calif), S-100 protein (COWS-100, dilution 1:500; Dako Corporation), Ki-67 (MIB-1, dilution 1:100; Dako Corporation), matrix metalloproteinase (MMP)-1 (41-1E5, dilution 1:100; Oncogene Research Products, Cambridge, Mass), MMP-2 (42-5D11, dilution 1:40; Oncogene Research Products), MMP-9 (56-2A4, dilution 1:60; Oncogene Research Products), tissue inhibitors of matrix proteinase (TIMP)-1 (102D1, dilution 1:50; Calbiochem, Darmstadt, Germany), TIMP-2 (T2-101, dilution 1:30; Quartett, Berlin, Germany), urokinase plasminogen activator (uPA) (polyclonal, dilution 1:100; DAKO, Hamburg, Germany), plasminogen activator inhibitor (PAI)-1 (polyclonal, dilution 1:200; Calbiochem), and cathepsin B (CatB) (polyclonal, dilution 1:50; Oncogene Research Products). Sections measuring 4-μm thick were treated with primary antibody, followed by staining with the avidin-biotin complex (Immunotech, Marseille, France) or alkaline phosphatase detection kit (Vector Laboratories, Burlingame, Calif). The antigen was retrieved with enzyme digestion for pancytokeratin, CAM5.2, and S-100 protein, and with microwave for HGF, vimentin, MIB-1, TIMP-1, TIMP-2, and PAI-1.

Expression of HGF and c-MET was graded semiquantitatively according to the number of positive cells: 0 indicates none, 1 indicates <10%, 2 indicates 10% to 50%, and 3 indicates >50%. Reactivity for cell differentiation markers (pancytokeratin, CAM5.2, vimentin, and S-100 protein) was graded semiquantitatively according to the number of positive cells: 0 indicates none,1+ indicates <80%, and 2+ indicates >80%.

The MIB-1-positive cells were counted in well-labeled areas, as determined by scanning at low magnification. The MIB-1 labeling index was determined as follows: 1) per 1000 tumor cells in the selected fields at ×400 magnification or 2) per all the tumor cells in 10 fields using the same magnification if there were <1000 cells.

The immunoreactivity for the proteinases, including MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, uPA, PAI-1, and CatB, was scored as previously described.41, 44, 45 The number of positive cells was semiquantitatively graded as: 0 indicates none, 1 indicates <10%, 2 indicates 10% to 50%, and 3 indicates >50%. Staining intensity was graded as: 0, negative; 1, faint; 2, moderate; and 3, strong. The final score was determined by adding these 2 indices (from 0 to 6).

Parts of the results for proteinases,45 MIB-1 labeling index,42, 46 and CAM5.245 were documented previously.

Statistical Analysis

Correlations between the expression of HGF/c-MET and patient age, MIB-1 labeling index, or proteinase scores were estimated using the Mann-Whitney U test. We compared the HGF/c-MET scores in 25 pairs of primary and recurrent lesions using the Student t test for paired data. The Fisher exact test was applied to define the correlation between the expression of HGF/c-MET and cell differentiation markers or other clinicopathologic parameters. The survival differences were tested using the Kaplan-Meier survival plot, and were evaluated using the Gehan-Wilcoxon method. A P value <.05 was considered to be statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

HGF and c-MET Scores

The HGF score was 1 in only a single primary skull base chordoma (Fig. 1B), and was scored as 0 in the remaining primary and recurrent lesions.

The staining pattern for c-MET was membranous or cytoplasmic (Fig. 1C). In primary tumors, the c-MET score was 0 in 14 lesions, 1 in 3 lesions, 2 in 4 lesions, and 3 in 25 lesions. In recurrent tumors, the c-MET score was 0 in 3 lesions, 1 in 4 lesions, 2 in 8 lesions, and 3 in 10 lesions. Consequently, 32 of 46 primary lesions (70.0%) and 22 of 25 recurrent lesions (88.0%) exhibited c-MET expression. Among these, 29 primary lesions (63.0%) and 18 recurrent lesions (72.0%) belonged to the higher c-MET expression group with a score of 2 or 3, respectively. The remaining 17 primary (37.0%) and 7 recurrent (28.0%) lesions belonged to the lower c-MET expression group with a score of 0 or 1. There were no significant differences noted with regard to the incidence of c-MET expression between primary and recurrent lesions.

No immunoreactivity for HGF and c-MET was observed in host bone marrow tissue.

Correlation between Expression of c-MET and Other Parameters

Patients with higher c-MET expression were, on average, 10 years younger (average ± standard deviation of 38.4 ± 14.9 years) than those with lower c-MET expression (48.0 ± 13.7 years), and the difference was found to be statistically significant (P = .037). There were no correlations noted between c-MET expression and sex, subsequent disease recurrence(s), nuclear pleomorphism, mitosis, apoptosis, necrosis, and bleeding.

The results of immunohistochemical analysis of cell differentiation markers, proteolytic enzymes, and MIB-1 labeling index are summarized in Table 1. There was a significant correlation noted between the expression of c-MET and CAM5.2 (Fig. 1D) in both primary (P = .002) and recurrent (P = .020) lesions. Pancytokeratin, vimentin, or S-100 protein expression did not appear to be correlated with c-MET expression. The expression of some proteinases was found to be correlated with c-MET expression. Recurrent lesions with higher c-MET expression presented with higher average scores of MMP-1, MMP-2, TIMP-1, uPA, and CatB, and the differences were found to be statistically significant or nearly significant for MMP-1 (P = .040), MMP-2 (P = .008), TIMP-1 (P = .052), and uPA (P = .073). However, in primary lesions, only uPA was found to be correlated with c-MET expression (P = .033). Proliferative ability evaluated with MIB-1 labeling index did not appear to be correlated with c-MET expression in either primary or recurrent lesions (Table 1).

Table 1. Correlation Between c-MET Score and CAM5.2 Score, MIB-1 LI, and Proteinase Scores in Primary and Recurrent Lesions
  c-MET Score in Primary Lesionsc-MET Score in Recurrent Lesions
Score 0 and 1 (n = 17)Score 2 and 3 (n = 29)PScore 0 and 1 (n = 7)Score 2 and 3 (n = 18)P
  • LI indicates labeling index; SD, standard deviation; MMP, matrix metalloproteinase; TIMP, tissue inhibitors of matrix proteinase; uPA, urokinase plasminogen activator; PAI, plasminogen activator inhibitor; CatB, cathepsin B.

  • *

    Statistically significant.

CAM5.2 score 0 and 1No. of patients1410 78 
 Score 2No. of patients319.002*010.020*
MIB-1 LIMean (SD)2.3 (3.8)3.1 (4.0).456.1 (4.7)9.1 (6.4).36
MMP-1Mean (SD)4.7 (1.1)4.8 (1.0).874.0 (1.0)4.8 (0.8).040*
MMP-2Mean (SD)4.2 (2.1)5.0 (1.3).303.7 (0.8)5.1 (1.2).008*
TIMP-1Mean (SD)3.9 (1.7)3.8 (2.0).942.7 (1.6)4.1 (1.8).052
TIMP-2Mean (SD)0.1 (0.5)0.2 (0.6).69001
uPAMean (SD)2.1 (1.7)3.2 (2.1).033*1.3 (2.2)3.3 (2.1).073
PAI-1Mean (SD)2.6 (1.4)1.6 (2.0).071.1 (1.5)0.9 (1.2).65
CatBMean (SD)0.8 (1.3)1.1 (1.6).460.6 (1.0)1.6 (1.7).16

Treatment and Prognosis

Brief clinical information regarding treatment was available for 38 patients. The lesions were initially excised in these patients. Eighteen patients received radiotherapy after the first surgery. Twenty-seven patients underwent multiple surgeries because of disease recurrence(s). Follow-up information was available for 29 patients; follow-up ranged from 7 to 185 months. The 5-year survival rate was 79.9% in patients with c-MET expression, and was 44.4% in those without c-MET expression (Fig. 2); there was a significant difference noted with regard to their survival rate (P = .032).

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Figure 2. Survival according to the expression of c-MET in skull base chordomas. Lesions with a higher level of c-MET expression were found to have a significantly better prognosis than those with lower c-MET expression.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The autocrine or paracrine HGF/c-MET signaling system appears to be correlated with proliferative ability, invasive ability, and advanced stage of disease, and plays a role in tumor progression in a wide spectrum of human malignancies. However, in some tumors, there is an inverse correlation between c-MET expression and tumor aggressiveness, and c-MET is considered to play a role in the early stages of neoplastic promotion. c-MET expression was frequently noted in well-differentiated tumor types, and was absent or observed only rarely in poorly differentiated types of thyroid cancer,31 breast carcinoma,33 and cholangiocarcinoma.32, 34 In addition, c-MET expression was found to be inversely correlated with vascular or perineural32 invasion and metastasis32, 47 in these tumors. Furthermore, patients with c-MET-positive lesions were found to have a more favorable prognosis than those with c-MET-negative lesions in some cancers.32–35 These differences in the role of HGF/c-MET signaling in tumors of various types may be due to the diverse effects of HGF on mitogenic,8–11 motogenic,5, 14 and morphogenic12, 13 functions.

In our previous study of malignant bone tumors, we found c-MET expression in 23% of osteosarcomas, 4.8% of malignant fibrous histiocytomas of bone, and 0% of Ewing sarcoma cases.36 By contrast, 17 of 18 lesions (94.4%) demonstrated c-MET expression in skull base and nonskull base chordomas.36 Weinberger et al.37 also reported that all 3 skull base chordomas in their study expressed c-MET. The incidence of c-MET expression in their small series of chordoma cases was obviously higher than that in bone tumors with a higher grade of malignancy. However, to our knowledge, the significance of HGF/c-MET expression with regard to invasive ability, proliferative ability, differentiation, and prognosis remains unknown in patients with skull base chordoma.

In the current study, we detected a high frequency of c-MET expression and only scarce immunoreactivity for HGF in a large series of primary and recurrent lesions of skull base chordoma. These results suggest the presence of a paracrine stimulation model, rather than an autocrine one, in this tumor.

Gain of chromosome 7q is one of the most common chromosome aberrations, and is considered an early event in chordoma genesis.48–50 The incidence of 7q gain was reported to amount to approximately 70% in what to our knowledge is the largest series reported to date.47 Conversely, proto-oncogene c-met is located at chromosome 7q31, and the incidence of c-MET expression in this study (70.0% in primary lesions) is quite similar to that of chromosome 7q gain. These findings indicate that c-MET expression via 7q amplification may play a role in the early stages of neoplastic promotion of skull base chordoma.

Chordoma is a unique bone tumor that retains both epithelial and mesenchymal characteristics. Histologically, chordoma is typically characterized by a syncytial arrangement, but to our knowledge a classification based on histologic differentiation in the conventional subtype does not exist. As previously reported in the literature,51 the staining score of low molecular weight cytokeratins (CAM5.2), corresponding to cytokeratins 8 and 18, varied. In addition, CAM5.2 expression was scored as 1+ in >50% of lesions in the current series. The clinical significance of divergent CAM5.2 expression remains controversial and to our knowledge the correlation between CAM5.2 expression and epithelial differentiation is unknown in chordoma. However, CAM5.2 expression may reflect the degree of epithelial differentiation in skull base chordoma. In the current study, there was a definite correlation noted between CAM5.2 and c-MET scores. Considering the finding that c-MET is expressed mainly in epithelial cells,11, 15–18 these results indicate that skull base chordomas with more epithelial differentiation express c-MET more strongly or suggest that those with less epithelial differentiation have decreased c-MET expression. This is quite similar to some carcinomas in which well-differentiated lesions strongly expressed c-MET, whereas poorly differentiated lesions demonstrated decreased c-MET expression.31–34

To our knowledge, there exists no widely accepted histologic grading system for skull base chordoma. Among histologic parameters, nuclear pleomorphism could be regarded as an indicator of aggressive biologic behavior, especially in nonskull base lesions.46 However, in the current study, we found no correlation between these histologic parameters, including nuclear pleomorphism, and c-MET expression. In addition, there was no apparent correlation between c-MET expression and MIB-1 labeling index in skull base chordoma. Moreover, the correlation between c-MET expression and proteinases was found to be weak in primary skull base chordoma. These results suggest a limited role for c-MET in the proliferative ability and invasive growth of skull base chordoma. Furthermore, patients with higher c-MET expression were found to have a significantly longer survival than those with lower c-MET expression. These findings indicate that the accumulation of c-MET protein does not necessarily correlate with disease progression in skull base chordoma, and a subsequent lack or loss of c-MET expression is considered to result in a more aggressive clinical behavior, leading to poor survival. This is in keeping with the correlation noted between c-MET expression and younger patient age in the current series, although patient age does not directly indicate the duration from tumor development to surgery. By contrast, recurrent lesions with higher c-MET expression exhibited an elevated expression of some proteinases. We believe that the discrepancy in the effect of HGF/c-MET signaling on proteinase expression between primary and recurrent lesions may be due to its multifunctional role during the neoplastic promotion of skull base chordoma, as was suggested for human carcinomas.

In conclusion, c-MET expression appears to be correlated with CAM5.2 expression and younger patient age, and a subsequent lack or loss of c-MET expression may cause aggressive behavior, leading to poor prognosis. However, with regard to the effect on the invasive ability of the tumor, further investigations are still necessary to clarify the differences in c-MET expression between primary and recurrent lesions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Ms Brigitte Roettger, Ms Carola Kuegler, Ms Claudia Miethke, and Ms Nadine Wiest, Department of Pathology, Faculty of Medicine at Magdeburg University for their skillful technical assistance. We also thank Dr. Kazuhiro Tanaka and Dr. Takashi Ikenoue of the Department of Orthopedic Surgery, Faculty of Medicine, Graduate School of Medical Sciences, Kyushu University, Japan for their helpful discussion, and Mr. Bernd Wuesthoff for editing the article.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES