Liver, 1991: 11, 272-277 Key words: epidermal growth factor; fibroblast growth factor; hepatocellular carcinoma; immunohistochemistry
Expression of epidermal growth factor and fibroblast growth factor in human hepatocellular carcinoma: an immunohistochemical study YOSHIHARU MOTOO’, NORIO SAWABU’ AND YASUNI
NAKANUMA’
‘Department of Internal Medicine, Cancer Research Institute and ‘Second Department of Pathology, School of Medicine, Kanazawa University, Kanazawa, Japan
ABSTRACT Expression of epidermal growth factor (EGF) and fibroblast growth factor (FGF) was examined in 56 patients with hepatocellular carcinoma (HCC) using an immunohistochemical method. EGF and FGF were expressed on carcinoma cells in 14 (25%) and 23 cases (41%), respectively. In the 23 FGF-positive cases, 11 cases were positive for both acidic and basic FGF, while 18 were positive for acidic FGF, and 16 were positive for basic FGF. In non-cancerous hepatic tissues, FGF was weakly positive in macrophages. hepatocytes and vascular endothelial cells in some cases, while EGF was totally negative. There were no significant correlations between the expression of EGF or FGF on carcinoma cells and the various clinicopathologic Factors examined. These data suggest that EGF and FGF are produced by human HCC cells in vivo. The roles of the expression of these growth factors in the development and progression of HCC remain only speculative. ~
Acceplrd,for publication 10 April 1991
Growth factors are well-known to play many physiologic roles in cell growth and differentiation, and are also involved in the development and progression of cancer (1). Among growth factors, epidermal growth factor (EGF) is important in the growth of normal as well as tumor cells. In fact, the level of EGF in urine has been reported to be increased in cancer patients (2). Fibroblast growth factor (FGF) is homologous with the product of oncogene hst-1 (3), and FGF is known to be a potent angiogenic factor (4). The FGF family consists of several factors with different properties. Among the FGF family, acid-
ic FGF (aFGF) and basic FGF (bFGF) have been the most extensively studied. We previously reported the presence of EGF receptor in human hepatoma cell lines as well as in human hepatocellular carcinoma (HCC) tissues (5). HCC is a hypervascular tumor, and is usually associated with liver cirrhosis or advanced hepatic fibrosis. It seems conceivable that FGF may also be very important in the development and progression of HCC. There have been, however, very few reports concerning EGF or FGF in human HCC tissues. In this study, we demonstrated immunohisto-
EGF AND FGF IN HEPATOMA
chemically that human HCC cells in vivo produce EGF and FGF. The relationship between the expression of these growth factors on HCC cells and various clinicopathologic factors was also investigated.
Material and methods Materials Fifty-six cases of HCC (43 males and 13 females, all adults) were studied. The diagnosis of HCC was made
273
pre-autopsy in all cases. Serum hepatitis B surface antigen (HBsAg) was positive in 10 of the 56 patients with HCC. Antibody to hepatitis C virus was not tested. All specimens were obtained at autopsy 2 4 h after death and were fixed in 10% neutral formalin. and embedded in paraffin. Deparaffinized sections were routinely stained with H & E, Azan stain and Gomori’s reticulin stain, and a pathologic diagnosis was made in each case. The gross features of HCC were classified as “diffuse”, “nodular” and “massive”, according to Eggel (6). The histology of HCC was classified as showing a “trabecular”, “pseudoglandular”, “compact” or “scirrhous” pattern according to the international classification (7). The degree of differen. .
A
Dilution of antibody
B
1 o2
103
1o4
105
Dilution of antibody
Fig. 1. A . Binding of anti-acidic F G F antibody to acidic FGF (0-0) and basic FGF (@-a), examined with enzymelinked immunosolvent assay (ELISA). E . Binding of anti-basic FG F antibody to basic FGF (0-0)and acidic FGF (0-0).examined with ELISA.
274
MOT00 ET AL.
tiation of cancer cells was graded from I to IV, according to Edmondson & Steiner (8).
Antibodies The mouse monoclonal antibody against human E G F was purchased from Wakunaga Pharmaceutical Company (Hiroshima, Japan). The rabbit polyclonal antibodies against a F G F and b F G F were raised in our laboratory. Briefly, synthetic peptides of the aminotcrminal oligopeptides of a F G F (1-1 I ) and b F G F (1-24) (Bachem, Inc., Torrance, CA, USA) were coupled with keyhole lympets hemocyanin (Sigma Chemical Company, St Louis, MO, USA) using glutaraldehyde. They were mixed with complete Freund's adjuvant and were then injected to rabbits (New Zealnd White) intradermally. Antisera to a F G F and b F G F showed high titers (Fig. I ) and were not cross-reactive. The anti-FGF antibodies did not react with EGF, transforming growth factor (TGF)-a, TGF-b, platelet-derived growth factor, insulin, insulin-like growth Factor-I, and interleukin-I (data not shown). Antisera were purified to IgG with ammonium sulfate and protein A affinity chromatography (Pharmacia, Uppsala, Sweden).
lmmunohistochemical method Five-pm paraffin sections were deparaffinized and incubated in methanol/H20, for the blocking of endogenous
peroxidase activity. After blocking nonspecific binding with normal serum, sections were incubated with antiE G F (diluted 150) or anti-FGF (diluted 1:200) at 4 C overnight, followed by incubation with biotinylated second antibody for 30 min, and avidin-biotin-peroxidase complex solution (Vectastain ABC kit, Vector Lab., Burlingame, CA, USA) for 20 min. Sections were treated with diaminobenzidine solution (Kyowa Medex, Tokyo. Japan), counterstained with hematoxylin, and mounted. The reaction specificity was examined using normal mouse or rabbit IgG, instead of primary antibodies.
Statistical analysis The chi-square test was used to analyze the data. Survival curves were constructed with the Kaplan-Meier method, and comparisons were made with the log-rank test. Statistical significance was defined as p < 0.05.
Results EGF was positively detected in HCC cells in 14 (25%) of the 56 cases of HCC, and reaction products were shown as granular patterns in the cytoplasm of the tumor cells (Fig. 2). Nuclei were negative for EGF. EGF-positive cells were distributed focally in the cancer nodules. EGF was not stained in the non-cancerous portions.
Fig. 2. Expression of E G F in the cytoplasm of HCC cells (arrows). The ABC method counterstained with hematoxylin. x400.
EGF AND FGF IN HEPATOMA
275
Fig. 3. Expression of basic FGF in the cytoplasm of HCC cells (arrows). The ABC method counterstained with hematoxylin, x 400.
FGF was positive in 23 (41%) of the 56 cases. Among the 23 FGF-positive cases, 18 cases were positive for aFGF, 16 were positive for bFGF, and 11 were positive for both. The staining pattern was also cytoplasmic and granular (Fig. 3), and nuclei were negative. The FGF-positive cells were focally distributed within the cancer nodules. Cirrhosis was seen in 53 of 56 patients with HCC. a F G F was positive in one of three non-cirrhotic patients. b F G F was negative in the three cases. In addition, FGF was also positive in macrophages, vascular endothelial cells and some hepatocytes in the non-cancerous portions. There was no difference in staining pattern between a F G F and bFGF. At least two sections were examined for each patient. Staining variability was seen among H C C cells in each section, but not between the sections. The correlations between the expression of these factors and various clinicopathologic factors were examined (Table I). There were no significant correlations between growth factor expression and the following clinicopathologic factors; serum alpha-fetoprotein levels, the differentiation grade (Edmondson-Steiner), presence of
hepatitis B surface antigen, presence of fibrous capsule, and macroscopic type of HCC (nodular, massive, or diffuse), metastasis including portal vein tumor thrombus or mean survival period after diagnosis.
Discussion In this study we demonstrated immunohistochemically the expression of E G F and F G F (acidic and basic) in human HCC cells. These factors were detected as fine granular staining patterns in the cytoplasm of the H C C cells, suggesting that E G F and FGF are produced by the human HCC cells themselves. The staining intensity and the number of positive cells varied, however, from cell to cell in the same cancer nodules, o r from one cancer nodule to another in the same case. SchulzeOsthoff et al. (9) also reported the heterogeneous staining of basic FGF in various tumors, not including hepatoma. These data suggest that HCC cells may be heterogeneous in FGF production. The possibility that the antibodies used do not recognize all the substances with EGF-like activity
276
MOT00 ET AL
Table 1 Relationship between the expression of growth factors and various clinicopathologic factors in 56 patients with HCC EGF
AFP (ngiml)
+
-
+
-
14
42
18
38
16
40
6 8
19 23
8 10
17 21
9 7
18 22
0 4 4 6
5 12 13 12
1 6 5 6
4
0 6 2 8
1 21 19 I
0 II 6
IV
0 9 5 0
N M D
9 4 1
26 13 3
9 7 2
26
2
9 33
3
5 400 52 255
55 10 10
Expression of epidermal growth factor and fibroblast growth factor in human hepatocellular carcinoma: an immunohistochemical study YOSHIHARU MOTOO’, NORIO SAWABU’ AND YASUNI
NAKANUMA’
‘Department of Internal Medicine, Cancer Research Institute and ‘Second Department of Pathology, School of Medicine, Kanazawa University, Kanazawa, Japan
ABSTRACT Expression of epidermal growth factor (EGF) and fibroblast growth factor (FGF) was examined in 56 patients with hepatocellular carcinoma (HCC) using an immunohistochemical method. EGF and FGF were expressed on carcinoma cells in 14 (25%) and 23 cases (41%), respectively. In the 23 FGF-positive cases, 11 cases were positive for both acidic and basic FGF, while 18 were positive for acidic FGF, and 16 were positive for basic FGF. In non-cancerous hepatic tissues, FGF was weakly positive in macrophages. hepatocytes and vascular endothelial cells in some cases, while EGF was totally negative. There were no significant correlations between the expression of EGF or FGF on carcinoma cells and the various clinicopathologic Factors examined. These data suggest that EGF and FGF are produced by human HCC cells in vivo. The roles of the expression of these growth factors in the development and progression of HCC remain only speculative. ~
Acceplrd,for publication 10 April 1991
Growth factors are well-known to play many physiologic roles in cell growth and differentiation, and are also involved in the development and progression of cancer (1). Among growth factors, epidermal growth factor (EGF) is important in the growth of normal as well as tumor cells. In fact, the level of EGF in urine has been reported to be increased in cancer patients (2). Fibroblast growth factor (FGF) is homologous with the product of oncogene hst-1 (3), and FGF is known to be a potent angiogenic factor (4). The FGF family consists of several factors with different properties. Among the FGF family, acid-
ic FGF (aFGF) and basic FGF (bFGF) have been the most extensively studied. We previously reported the presence of EGF receptor in human hepatoma cell lines as well as in human hepatocellular carcinoma (HCC) tissues (5). HCC is a hypervascular tumor, and is usually associated with liver cirrhosis or advanced hepatic fibrosis. It seems conceivable that FGF may also be very important in the development and progression of HCC. There have been, however, very few reports concerning EGF or FGF in human HCC tissues. In this study, we demonstrated immunohisto-
EGF AND FGF IN HEPATOMA
chemically that human HCC cells in vivo produce EGF and FGF. The relationship between the expression of these growth factors on HCC cells and various clinicopathologic factors was also investigated.
Material and methods Materials Fifty-six cases of HCC (43 males and 13 females, all adults) were studied. The diagnosis of HCC was made
273
pre-autopsy in all cases. Serum hepatitis B surface antigen (HBsAg) was positive in 10 of the 56 patients with HCC. Antibody to hepatitis C virus was not tested. All specimens were obtained at autopsy 2 4 h after death and were fixed in 10% neutral formalin. and embedded in paraffin. Deparaffinized sections were routinely stained with H & E, Azan stain and Gomori’s reticulin stain, and a pathologic diagnosis was made in each case. The gross features of HCC were classified as “diffuse”, “nodular” and “massive”, according to Eggel (6). The histology of HCC was classified as showing a “trabecular”, “pseudoglandular”, “compact” or “scirrhous” pattern according to the international classification (7). The degree of differen. .
A
Dilution of antibody
B
1 o2
103
1o4
105
Dilution of antibody
Fig. 1. A . Binding of anti-acidic F G F antibody to acidic FGF (0-0) and basic FGF (@-a), examined with enzymelinked immunosolvent assay (ELISA). E . Binding of anti-basic FG F antibody to basic FGF (0-0)and acidic FGF (0-0).examined with ELISA.
274
MOT00 ET AL.
tiation of cancer cells was graded from I to IV, according to Edmondson & Steiner (8).
Antibodies The mouse monoclonal antibody against human E G F was purchased from Wakunaga Pharmaceutical Company (Hiroshima, Japan). The rabbit polyclonal antibodies against a F G F and b F G F were raised in our laboratory. Briefly, synthetic peptides of the aminotcrminal oligopeptides of a F G F (1-1 I ) and b F G F (1-24) (Bachem, Inc., Torrance, CA, USA) were coupled with keyhole lympets hemocyanin (Sigma Chemical Company, St Louis, MO, USA) using glutaraldehyde. They were mixed with complete Freund's adjuvant and were then injected to rabbits (New Zealnd White) intradermally. Antisera to a F G F and b F G F showed high titers (Fig. I ) and were not cross-reactive. The anti-FGF antibodies did not react with EGF, transforming growth factor (TGF)-a, TGF-b, platelet-derived growth factor, insulin, insulin-like growth Factor-I, and interleukin-I (data not shown). Antisera were purified to IgG with ammonium sulfate and protein A affinity chromatography (Pharmacia, Uppsala, Sweden).
lmmunohistochemical method Five-pm paraffin sections were deparaffinized and incubated in methanol/H20, for the blocking of endogenous
peroxidase activity. After blocking nonspecific binding with normal serum, sections were incubated with antiE G F (diluted 150) or anti-FGF (diluted 1:200) at 4 C overnight, followed by incubation with biotinylated second antibody for 30 min, and avidin-biotin-peroxidase complex solution (Vectastain ABC kit, Vector Lab., Burlingame, CA, USA) for 20 min. Sections were treated with diaminobenzidine solution (Kyowa Medex, Tokyo. Japan), counterstained with hematoxylin, and mounted. The reaction specificity was examined using normal mouse or rabbit IgG, instead of primary antibodies.
Statistical analysis The chi-square test was used to analyze the data. Survival curves were constructed with the Kaplan-Meier method, and comparisons were made with the log-rank test. Statistical significance was defined as p < 0.05.
Results EGF was positively detected in HCC cells in 14 (25%) of the 56 cases of HCC, and reaction products were shown as granular patterns in the cytoplasm of the tumor cells (Fig. 2). Nuclei were negative for EGF. EGF-positive cells were distributed focally in the cancer nodules. EGF was not stained in the non-cancerous portions.
Fig. 2. Expression of E G F in the cytoplasm of HCC cells (arrows). The ABC method counterstained with hematoxylin. x400.
EGF AND FGF IN HEPATOMA
275
Fig. 3. Expression of basic FGF in the cytoplasm of HCC cells (arrows). The ABC method counterstained with hematoxylin, x 400.
FGF was positive in 23 (41%) of the 56 cases. Among the 23 FGF-positive cases, 18 cases were positive for aFGF, 16 were positive for bFGF, and 11 were positive for both. The staining pattern was also cytoplasmic and granular (Fig. 3), and nuclei were negative. The FGF-positive cells were focally distributed within the cancer nodules. Cirrhosis was seen in 53 of 56 patients with HCC. a F G F was positive in one of three non-cirrhotic patients. b F G F was negative in the three cases. In addition, FGF was also positive in macrophages, vascular endothelial cells and some hepatocytes in the non-cancerous portions. There was no difference in staining pattern between a F G F and bFGF. At least two sections were examined for each patient. Staining variability was seen among H C C cells in each section, but not between the sections. The correlations between the expression of these factors and various clinicopathologic factors were examined (Table I). There were no significant correlations between growth factor expression and the following clinicopathologic factors; serum alpha-fetoprotein levels, the differentiation grade (Edmondson-Steiner), presence of
hepatitis B surface antigen, presence of fibrous capsule, and macroscopic type of HCC (nodular, massive, or diffuse), metastasis including portal vein tumor thrombus or mean survival period after diagnosis.
Discussion In this study we demonstrated immunohistochemically the expression of E G F and F G F (acidic and basic) in human HCC cells. These factors were detected as fine granular staining patterns in the cytoplasm of the H C C cells, suggesting that E G F and FGF are produced by the human HCC cells themselves. The staining intensity and the number of positive cells varied, however, from cell to cell in the same cancer nodules, o r from one cancer nodule to another in the same case. SchulzeOsthoff et al. (9) also reported the heterogeneous staining of basic FGF in various tumors, not including hepatoma. These data suggest that HCC cells may be heterogeneous in FGF production. The possibility that the antibodies used do not recognize all the substances with EGF-like activity
276
MOT00 ET AL
Table 1 Relationship between the expression of growth factors and various clinicopathologic factors in 56 patients with HCC EGF
AFP (ngiml)
+
-
+
-
14
42
18
38
16
40
6 8
19 23
8 10
17 21
9 7
18 22
0 4 4 6
5 12 13 12
1 6 5 6
4
0 6 2 8
1 21 19 I
0 II 6
IV
0 9 5 0
N M D
9 4 1
26 13 3
9 7 2
26
2
9 33
3
5 400 52 255
55 10 10
