Objective: Hepatocyte growth factor (HGF) has been reported the cause of many biological events, including cell proliferation, movement, invasiveness, morphogenesis, and angiogenesis. Elevated hepatocyte growth factor content in tumor tissue was reported to predict a more aggressive biology in non–small cell lung cancer patients. However, there is still limited knowledge about the role of HGF in breast cancer. This study was designed with the aim to elucidate the possible relationship between the preoperative circulating soluble HGF and breast cancer.

Materials and Methods: One hundred twenty-four consecutive patients with invasive breast cancer undergoing surgery were prospectively included and evaluated. Venous blood samples were collected before the surgery. Sera were obtained by centrifugation and stored at −70°C until assayed. The control group consisted of 35 patients with benign breast tumor (20 with fibrocystic disease and 15 with fibroadenoma). Serum concentrations of soluble HGF were measured by the quantitative sandwich enzyme immunoassay technique. The data on primary tumor staging, age, estrogen receptor status, lymph node status, distant metastases status, histologic grading, and tumor-node-metastasis (TNM) staging were reviewed and recorded.

Results: The mean value of serum soluble HGF in patients with invasive breast cancer was 529.05 ± 123.33 pg/mL and that of control group was 343.00± 31.03 pg/mL and the difference was significant (P < 0.001). Furthermore, there were significantly higher serum levels of soluble HGF in patients with negative estrogen receptor (P = 0.035), in patients with poorer differentiated tumor (P < 0.001), in patients with more advanced primary tumor staging (P < 0.001), in patients with more advanced lymph node status (P < 0.001), in patients with distant metastases (P < 0.001), and in patients with more advanced TNM staging (P < 0.001). In multivariate analysis by the multiple linear regression method, TNM staging (P < 0.001) seemed an independent factor regarding the significant higher serum levels of soluble HGF.

Conclusion: Patients with more advanced TNM staging were shown to have higher serum soluble HGF. Thus, preoperative serum soluble HGF levels might reflect the severity of invasive breast cancer and deserve further evaluation.

Hepatocyte growth factor (HGF),which is known to be identical to scatter factor, has been reported the cause of many biological events, including cell proliferation (1), movement (2), invasiveness (3, 4), morphogenesis (5), and angiogenesis (6). Hepatocyte growth factor is found in many organs, including the mammary gland, lung, kidney, and liver (7, 8). Elevated hepatocyte growth factor content in tumor tissue was reported to predict a more aggressive biology in non–small cell lung cancer patients (9). However, there is still limited knowledge about the role of HGF in breast cancer. The evaluation of the possible outcome of the patient with breast cancer is important for planning optional treatment. Because no single prognostic factor can determine the whole status of a patient with breast cancer, the physician must consider all available prognostic data. This study was designed with the aim to investigate any correlation between the preoperative circulating hepatocyte growth factor and the clinicopathologic features and to evaluate the possible prognostic significance of the preoperative circulating hepatocyte growth factor in breast cancer.

From November 1998 to October 2001, 124 consecutive patients with invasive breast cancer were included in this study. All the patients met the following criteria: (a) having been diagnosed as having primary invasive breast cancer, (b) having no clinical manifestation of infection, (c) having received no immunomodulatory agents during the previous 3 weeks, (d) having received no blood transfusion during the previous 3 weeks, (e) having no known liver dysfunction, (f) having no known lung or renal dysfunction, and (g) having no other known malignancy. Venous blood samples were collected before the surgery. Sera were obtained by centrifugation and stored at −70°C until assayed. All 124 patients were women ages 31 to 84 years (mean, 50.5 years). All patients underwent modified radical mastectomy except patients with stage IV and the diagnosis of breast cancer was confirmed by histologic examination. Invasive breast cancer was defined as carcinoma, regardless of origin (duct or lobule), with invasion to or beyond the basement membrane (10). The data of primary tumor staging, age, estrogen receptor status, lymph node and metastasis status, histologic grading, and tumor-node-metastasis (TNM) staging were collected. Thorough physical examination, chest radiography, level of serum alkaline phosphatase, and mammogram were the preoperative routine for all patients. Bone scan and abdominal ultrasonography were done for all patients with provisional clinical stage III or above to rule out the presence of distant metastases. Regardless of any provisional clinical stage, all patients with elevated serum alkaline phosphatase special complaints such as bone pain or any specific findings indicating the possibility of distant metastases such as hepatomegaly also underwent bone scan and abdominal ultrasonography to detect if there was distant metastases. All of the cancer were graded according to the criteria described by Bloom and Richard (11). Estrogen receptor status was determined by immunohistochemical staining method (12-17). Thirty-five patients with benign breast tumor (20 patients with fibrocystic disease and 15 patients with fibroadenoma) were used as control group.

This assay (R&D Systems, Inc. Minneapolis, MN) employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for HGF has been precoated onto a microplate. Standards and samples are pipetted into the wells and any HGF present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for HGF is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HGF bound in the initial step. The color development is stopped and the intensity of the color is measured. The minimum detectable dose of HGF is typically <40 pg/mL.

Statistical Analysis

The Student's t test was used to assess the significance of difference in the levels of HGF between the patient group and control group. The following clinicopathologic variables were first entered into the univariate analysis by Student's t test or ANOVA test: primary tumor staging, age, estrogen receptor status, lymph node status, distant metastases status, histologic grading, and TNM staging. These clinicopathologic variables were then assessed by the multiple linear regression method. P < 0.05 was accepted as significant. Results in pg/mL were expressed as the mean ± SD.

The mean value of serum soluble HGF in patients with invasive breast cancer was 529.1 ± 123.3 pg/mL and that of control group was 343.0 ± 31.0 pg/mL and the difference was significant (P < 0.001). Furthermore (Table 1), there were significantly higher serum levels of soluble HGF in patients with negative estrogen receptor (P = 0.035), in patients with poorer differentiated tumor (P < 0.001), in patients with more advanced primary tumor staging (P < 0.001), in patients with more advanced lymph node status (P < 0.001), in patients with distant metastases (P < 0.001), and in patients with more advanced TNM staging (P < 0.001). In multivariate analysis by the multiple linear regression method, TNM staging (P < 0.001) seemed an independent factor regarding the significant higher serum levels of soluble HGF.

Table 1.

Serum concentrations of HGF in relation to clinicopathologic variables

nMean value of HGF (pg/mL)P
Age (y)    
    <50 65 535.5 ± 142.2 0.545 
    ≥50 59 522.0 ± 99.2  
Estrogen receptor    
    Negative 49 557.9 ± 135.7 0.035 
    Positive 75 510.2 ± 111.5  
Primary tumor staging    
    T1 14 367.7 ± 56.9 <0.001 
    T2 70 491.0 ± 75.4  
    T3 11 600.5 ± 119.7  
    T4 29 672.0 ± 79.8  
Lymph node status    
    No 58 454.0 ± 77.5 <0.001 
    N1 33 516.7 ± 97.7  
    N2 33 673.2 ± 80.3  
Distant metastases    
    Absent 114 505.2 ± 96.8 <0.001 
    Present 10 801.2 ± 25.5  
TNM staging    
    I 11 343.4± 31.6 <0.001 
    II 66 463.9± 34.0  
    III 37 627.0 ± 26.4  
    IV 10 801.2 ± 25.5  
Histologic grading (differentiation)    
    Well 45 438.9 ± 65.0 <0.001 
    Moderately 56 536.6 ± 100.2  
    Poor 23 686.1 ± 92.8  
nMean value of HGF (pg/mL)P
Age (y)    
    <50 65 535.5 ± 142.2 0.545 
    ≥50 59 522.0 ± 99.2  
Estrogen receptor    
    Negative 49 557.9 ± 135.7 0.035 
    Positive 75 510.2 ± 111.5  
Primary tumor staging    
    T1 14 367.7 ± 56.9 <0.001 
    T2 70 491.0 ± 75.4  
    T3 11 600.5 ± 119.7  
    T4 29 672.0 ± 79.8  
Lymph node status    
    No 58 454.0 ± 77.5 <0.001 
    N1 33 516.7 ± 97.7  
    N2 33 673.2 ± 80.3  
Distant metastases    
    Absent 114 505.2 ± 96.8 <0.001 
    Present 10 801.2 ± 25.5  
TNM staging    
    I 11 343.4± 31.6 <0.001 
    II 66 463.9± 34.0  
    III 37 627.0 ± 26.4  
    IV 10 801.2 ± 25.5  
Histologic grading (differentiation)    
    Well 45 438.9 ± 65.0 <0.001 
    Moderately 56 536.6 ± 100.2  
    Poor 23 686.1 ± 92.8  

The evaluation of the possible outcome of the patient with breast cancer is important for planning optional treatment. Because no single prognostic factor can determine the whole status of a patient with breast cancer, the physician must consider all available prognostic data. This study was conducted to evaluate the correlation of circulating soluble hepatocyte growth factor with clinicopathologic variables and its possible prognostic value with the hope to find additional meaningful information for making a treatment decision. In this study, serum was chosen for measurement of hepatocyte growth factor because serum is easily available and sufficient for an objective analysis. The data could be achieved preoperatively and could be useful to an optimal preoperative planning. This method for measurement of hepatocyte growth factor is feasible, reproducible, inexpensive, and widely available; the serum levels of hepatocyte growth factor have already been shown to be of prognostic value in other malignancy (9). Although semiquantitative analysis by immunohistochemical staining of tumor specimen has been claimed to be a useful method to detect a tumor mark (18), this method indeed has several drawbacks. First, although semiquantitative evaluation is sufficient to differentiate negative versus positive reaction, it is sometimes not accurate enough to evaluate the intermediate patterns of staining. Second, the possibility of heterogeneity within tumor specimens might cause different results. Finally, different types of antibodies and procedures were used in different series and could lead to different results. The choice of serum for a quantitative analysis in this study could possibly avoid the abovementioned disadvantages of a semiquantitative analysis by immunohistochemical staining. Only patients with invasive breast carcinoma was included, because noninvasive carcinoma usually carries a quite different clinical course. Patients with clinical infection were excluded because infection itself would theoretically cause an altered concentration of serum cytokine. In addition, immunomodulatory agents and blood transfusions were avoided to allow for an objective, accurate assay of cytokines. Patients with known liver, lung, or renal dysfunction were also excluded to avoid the possible interfering factors. Patients with known malignancy other than breast cancer were excluded since the increase in the serum HGF was found in patients with other neoplasms including gastric cancer, lymphomas, and leukemias (19).

HGF is found to be involved in carcinogenesis. Jeffers et al. (20) reported that cotransfection of HGF and c-met was able to induce morphologic transformation in vitro and tumorigenicity in vivo in a nontumorigenic mouse cell line C127. In the bladder cancer line NBT-II, transfection of HGF upgraded the invasive phenotype and growth rate of these cells (21). The mammary stroma is reported to exert a paracrine influence on the neighboring epithelial cell population (22). It has been noted that an intimate interaction may exist in fibroblast HGF expression between epithelial and mesenchymal compartments in human breast cancer depending on the in vivo conditions (23). It was reported that HGF receptor is widely distributed in various epithelial cells including tumor cells but obviously not in mesenchymal cells (24). On the other hand, HGF production was found in the stromal component but not in the epithelial component of the breast (8, 23). Because it has been reported that HGF is a modulator of epithelial cell proliferation and motility for a broad spectrum of cell types (7, 25), it is tempting to speculate that HGF originating from breast stromal cells may play a crucial role in facilitating breast cancer cell invasion and metastasis.

In multivariate analysis by the multiple linear regression method, TNM staging (P < 0.001) seemed an independent factor regarding the significant higher serum levels of soluble HGF. Based on the results, the higher preoperative level of serum HGF is shown closely related to a more advanced TNM stage (Table 1). Thus, the preoperative level of serum HGF may reflect the severity of invasive breast cancer and may be useful to pick up higher risk patients for more aggressive treatment. It is worthwhile to have further investigation by larger group of patients with longer follow-up to achieve more substantial conclusion.

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.

1
Singh-Kaw P, Zarnegar R, Siegfried JM. Stimulatory effects of hepatocyte growth factor on normal and neoplastic human epithelial cells.
Am J Physiol
1995
;
268
:
L1012
–20.
2
Rosen EM, Knesel J, Goldberg ID, Jin L, et al. Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumor.
Int J Cancer
1994
;
57
:
706
–14.
3
Weidner KM, Behrens J, Vandekerckhove J, et al. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.
J Cell Biol
1990
;
111
:
2097
–108.
4
Nakamura T, Matsumoto K, Kiritoshi A, et al. Induction of hepatocyte growth factor in fibroblasts by tumor-derived factors affects invasive growth of tumor cells: in vitro analysis of tumor-stromal interactions.
Cancer Res
1997
;
57
:
3305
–13.
5
Montesan R, Matsumoto K, Nakamura T, et al. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.
Cell
1991
;
67
:
901
–8.
6
Grant DS, Kleinman HK, Goldberg ID, et al. Scatter factor induces blood vessels formation in vivo.
Proc Natl Acad Sci U S A
1993
;
90
:
1937
–41.
7
Igawa T, Kanda S, Kanetake H, et al. Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells.
Biochem Biophys Res Commun
1991
;
174
:
831
–8
8
Yamashita J, Ogawa M, Beppu T. Immunoreactive hepatocyte growth factor is present in tissue extracts from human breast cancer but not in conditioned medium of human breast cancer cell lines.
Res Commun Chem Pathol Pharmacol
1993
;
82
:
249
–52.
9
Siegfried JM, Weissfeld LA, Luketich JD, et al. The clinical significance of hepatocyte growth factor for non-small cell lung cancer.
Ann Thorac Surg
1998
;
66
:
1915
–8.
10
The World Health Organization histological typing of breast tumor, 2nd ed.
Am J Clin Pathol
1982
;
78
:
808
–16.
11
Bloom HJG, Richardson WW. Histological grading and prognosis in breast cancer.
Br J Cancer
1957
;
11
:
359
–77.
12
Shyr-Ming Sheen-Chen, Wei-Jen Chen, Hock-Liew Eng, Fong-Fu Chou. Serum concentration of tumor necrosis factor in patients with breast cancer.
Breast Cancer Res Treat
1997
;
43
:
211
–5.
13
Shyr-Ming Sheen-Chen, Hock-Liew Eng, Fong-Fu Chou, Wei-Jen Chen. The prognostic significance of proliferating cell nuclear antigen in patients with lymph node-positive breast cancer.
Arch Surg
1997
;
132
:
264
–7.
14
Shyr-Ming Sheen-Chen, Wei-Jen Chen, Hock-Liew Eng, Fong-Fu Chou. Serum-soluble interleukin-2 receptor concentrations in patients with breast cancer: a preliminary report.
Chang Gung Med J
1998
;
21
:
133
–8.
15
Shyr-Ming Sheen-Chen, Fong-Fu Chou, Hock-Liew Eng, Wei-Jen Chen. An evaluation of the prognostic significance of HLA-DR expression in axillary-node-negative breast cancer.
Surgery
1994
;
116
:
510
–5.
16
Shyr-Ming Sheen-Chen, Hock-Liew Eng, Chih-Wei Sheen, et al. Serum levels of circulating intercellular adhesion molecule-1 in patients with breast cancer.
Anticancer Res
1997
;
17
:
282
–36.
17
Shyr-Ming Sheen-Chen, Wei-Jen Chen, Hock-Liew Eng, et al. Evaluation of the prognostic value of serum soluble CD44 in patients with breast cancer.
Cancer Invest
1999
;
17
:
581
–5.
18
Tempfer C, Losch A, Heinzl H, et al. Prognostic value of immunohistochemically detected CD44 isoforms CD44 V5, CD44 V6 and CD44 V7,8 in human breast cancer.
Eur J Cancer
1996
;
32A
:
2023
–5.
19
Taniguchi T, Kitamura M, Arai K, et al. Increase in the circulating level of hepatocyte growth factor in gastric cancer patients.
Br J Cancer
1997
:
75
:
673
–7.
20
Jeffer M, Rong S, Anver M, Woude GFV. Autocrine hepatocyte growth factor/scatter factor-met signaling induces transformation and the invasive/metastatic phenotype in C127 cells.
Oncogene
1996
;
13
:
853
–61.
21
Bellusci S, Mones G, Gaudino G, et al. Creation of an hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity.
Oncogene
1994
;
9
:
1091
–9.
22
Oka T, Yoshimura M. Paracrine regulation of mammary gland growth.
J Clin Endocrinol Metab
1986
;
15
:
79
–97.
23
Seslar SP, Nakamura T, Stephen WB. Regulation of fibroblast hepatocyte growth factor/scatter factor expression by human breast carcinoma cell lines and peptide growth factors.
Cancer Res
1993
;
53
:
1233
–8.
24
Tajima H, Matsumoto K, Nakamura T. Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines.
FEBS Lett
1991
;
291
:
229
–32.
25
Matsumoto K, Tajima H, Nakamura T. Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth.
Biochem Biophys Res Commun
1991
;
176
:
45
–51.