645 Q 1992 The Japanese Society of Pathology
Immunohistochemical Study of Transforming Growth Factor p, Fibronectin, and Fibronectin Receptor in lnvasive Mammary Carcinomas Koji Oda’, Sadaaki Hori2, Hitoshi Noh2, R. Yoshiyuki Osamural, Yutaka Tokuda3, Mitsuharu Kubota3, and Tomoo Tajima3
Thirty-one cases of mammary carcinoma were examined immunohistochemically for the expression of transforming growth factor (TGF) /3, fibronectin (FN) and fibronectin receptor (FNR) in order to clarify the reason for the reported relationship between TGFp expression and a high incidence of lymph node metastasis. It was revealed that TGF 3/ expression is closely related to the expression of FN, an intercellular matrix protein, and its cellular receptor FNR, one of the integrins. The interaction between FN and FNR in a tumor is considered to form the basis of the invasive nature of carcinoma cells. Thus, it is suggested that TGF /3 expression in carcinoma cells induces the interaction between FN and FNR, which may lead to carcinomatous invasion resulting in lymph nodal metastasis. Aeta Pathol Jpn 4 2 : 6 4 5 6 5 0 , 1 9 9 2 . Key words : Transforming growth factor p, Mammary carcinoma, Growth factor
Transforming growth factor p (TGFp1) is a dimeric polypeptide with a molecular mass of 25 kDa, which was first reported by Tadaro et a/. as a growth factor that allows the anchorage independent growth of normal fibroblasts (1). This original definition of the activity of TGFp has now been expanded to include actions on many tissue and cell types including not only mesenchymal cells, but also epithelial cells, endothelial cells, blood cells, lymphocytes and neural cells (2, 3). With regard to functional aspects, it is well known that TGFp has multifunctional biological activities besides its conReceived January 17, 1992. Accepted for publication May 18, 1992. ‘Department of Pathology, Tokai University, School of Medicine, Kanagawa. ‘Department of Diagnostic Pathology, Tokai University Hospital, Konagawa. 3Department of Surgery, Tokai University, School of Medicine, Kanagawa. Mailing address: Koji Oda, Department of Pathology, Tokai University, School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1 1, Japan.
ventional regulatory effects on cell proliferation and differentiation (4). Among these, its effects on the synthesis of intercellular matrix proteins and the “integrin” cell receptors for them have been a focus of interest in attempts to understand the mechanisms of tumor invasion and metastasis (5-8). Several human mammary carcinoma cell lines are known t o secrete TGF p (9) and it has also been reported that TGF /3 shows growth-inhib itory effects on breast cancer cells in vitro (10-12). In human mammary tissue in vivo, on the other hand, Travers et a/. (13) have shown that T G F p messenger RNA is more abudant in carcinoma than in non-malignant breast tissue from studies of 77 biopsied breast specimens. We have also reported stronger immunohistochemical expression of T G F p in invasive breast carcinomas with lymph nodal metastasis than in those without lymph nodal metastasis (14, 15). In the present immunohistochemical study, the effects of T G F p on the induction of the extracellular matrix protein fibronectin (FN), and fibronectin receptor (FNR), one of the integrins, were examined. FN and FNR are known to play significant roles in tumor invasion and metastasis(4, 16, 17). The effects of T G F p in determining the histological types of breast carcinoma are also discussed.
MATERIALS AND METHODS Thirty -one surgically resected breast carcinomas from Japanese women were collected at the Division of Diagnostic Pathology, Tokai University Hospital. The clinical data and the specimen sources are shown in Table 1. The histological types of the breast carcinomas were determined according to the histological typing of breast tumors by the Japan Mammary Cancer Society (18). Individual cases sometimes expressed various histological types, especially with regard to the desmoplas-
646
Expression of TGFp in Human Mammary Carcinomas (Oda et a/.)
tic reaction. For this reason, among 3 1 cases of invasive ductal carcinoma, there were 12 foci showing a strong desmoplastic reaction (scirrhous lesions ; more than 50% of the tumor replaced by fibrous tissue) and 24 foci without a desmoplastic reaction (non-scirrhous lesions). All materials were frozen immediately in 0. C. T. compound and kept frozen for immunohistochemical study at -70 centigrade. Frozen sections 6 p m thick for immunohistochemistry were cut and fixed in 4% paraformaldehyde at room temperature for 30 min. The indirect immunoperoxidase method was performed using anti-TGFp1 polyclonal antibody (R & D System, Inc., U.S.A.), anti-FN monoclonal antibody (Transforming Research, Inc., U.S.A.) and anti-FNR monoclonal antibody (Telios Pharmaceuticals, Inc., U.S.A.). Sites of peroxidase reactivity were visualized using 25% diaminobenzidine, pH 7.6. The sections were counterstained with 5 % methyl green, pH 4.0. lmmunoreactivity of TGFp was divided into three grades; negative (-), positive (+) and strongly positive ( i t ) , according to the staining intensity of the
majority (more than 60%) of the cancer cells in the lesion. Fibroblasts in the non-neoplastic breast tissue stroma around the carcinoma were used as the standard for intensity of the TGFp staining. That is, in the cases with positive (+) TGFp staining, more than 60% of the examined cancer cells showed a TGF p staining intensity comparable to that of stromal fibroblasts. Strong positivity ( i t ) was defined as TGF p staining which was more intense than that of stromal fibroblasts in the majority of the cancer cells. lmmunoreactivities for FN and FNR were divided into two groups; negative (-) and positive (+), according to the imrnunoreactivity of each antigen in the majority (more than 60%) of the cancer cells.
RESULTS 1. lmmunoreactivity of TGFP, FN and FNR in 31 invasive carcinomas The results of the immunohistochemical staining of TGFp, FN and FNR are shown in Table 1. TGFp stain-
Table 1. Details of the Present Cases and lmmunohistochemistry of Mammary Carcinomas Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Patient age
51 45 41 54 46 71 52 49 40 69 50 32 47 65 60 48 53 43 33 72 43 49 43 47 44 89 44 44 49 33 68
Histology ST Sci ST MED Sci MUC Sci Sci PT ST ST PT Sci
ST ST PT PT ST
SQ MUC Sci ST Sci Sci ST Sci PT PT Sci PT ST
Maximum size (cm)
2.1 1.2 3.0 2.0 8.0 2.5 3.0 2.5 1.6 2.2 2.5 2.5 1.8 1.1 2.1 3.5 3.5 3.5 1.6 2.0 4.5 2.0
4.2 1.5 3.2 3.0 2.0 4.0 1.8 5.0 2.5
Lymph node metastasis negative negative negative positive positive positive positive positive negative negative negative positive negative negative positive positive positive negative negative negative positive positive positive positive positive positive negative negative negative positive positive
TGF ~3 S-lesion NS-lesion
/ it
/ it it
/ it it it
/ / / it
9 it
/ / / it
/
+
/ it
/ / / / /
+ +
/
+ + + it
/ it
+ / + + + + / / /
+ ++ it + + + + + + + + +
/ /
it
S-lesion
/
-
/
+ +
/
+ +
/ / / /
+ + +
FNR NS-lesion -
+ + / + -
/
+ + -
/ / /
/ /
-
+
+ + + +
/
/
+
/
/ / / / / /
-
+
/
PT : Papillo-tubular carcinoma, ST: Solid-tubular carcinoma, Sci : Scirrhous carcinoma, MUC : Mucinous carcinoma, MED : Medullary carcinoma, SQ: Squamous cell carcinoma (Japan Mammary Cancer Society).
-
+
-
-
/ /
-
Acta Pathologica Japonica 42 (9): 1992
ing was positive in all 31 cases of mammary carcinoma. TGFp was localized mainly in the cytoplasm of the cancer cells as well as in the stromal fibroblasts. The staining intensity of the carcinoma cells shown in Fig. l a was interpreted as strongly positive (+). In Fig. 2a, the staining intensity was interpreted as positive (+). Thirteen (41.9%) cases in the present study demonstrated strongly positive ( 8 )staining and 18 (58.1%) revealed positive (+) staining for TGFp. FN immunoreactivity was detected in the stroma in all 31 cases (Fig. 3b). FNR immunoreactivity was present mainly in the cancer cell membrane. The FNR staining was positive (+) in 17 (54.8%) and negative in 14 (45.2%) of the 33 carcinomas.
64 7
1 7 cases with only NS-lesions, only 2 revealed strongly positive TGFp expression. Among the 7 cases with solely S-lesions, 5 demonstrated strongly positive TGF p expression. Among the 7 cases with both S- and NSlesions, 6 demonstrated strongly positive TGF p expression in the S-lesions and 5 showed positive TGFp expression in the NS-lesions. Various degrees of TGFp expression in one case was noted in 4 cases, where the Slesion demonstrated strong TGF p expression and the NS-lesion revealed positive TGF p expression. Strongly positive TGF B expression was significantly more f r e quent in S-lesions than in NS-lesions (p
Immunohistochemical Study of Transforming Growth Factor p, Fibronectin, and Fibronectin Receptor in lnvasive Mammary Carcinomas Koji Oda’, Sadaaki Hori2, Hitoshi Noh2, R. Yoshiyuki Osamural, Yutaka Tokuda3, Mitsuharu Kubota3, and Tomoo Tajima3
Thirty-one cases of mammary carcinoma were examined immunohistochemically for the expression of transforming growth factor (TGF) /3, fibronectin (FN) and fibronectin receptor (FNR) in order to clarify the reason for the reported relationship between TGFp expression and a high incidence of lymph node metastasis. It was revealed that TGF 3/ expression is closely related to the expression of FN, an intercellular matrix protein, and its cellular receptor FNR, one of the integrins. The interaction between FN and FNR in a tumor is considered to form the basis of the invasive nature of carcinoma cells. Thus, it is suggested that TGF /3 expression in carcinoma cells induces the interaction between FN and FNR, which may lead to carcinomatous invasion resulting in lymph nodal metastasis. Aeta Pathol Jpn 4 2 : 6 4 5 6 5 0 , 1 9 9 2 . Key words : Transforming growth factor p, Mammary carcinoma, Growth factor
Transforming growth factor p (TGFp1) is a dimeric polypeptide with a molecular mass of 25 kDa, which was first reported by Tadaro et a/. as a growth factor that allows the anchorage independent growth of normal fibroblasts (1). This original definition of the activity of TGFp has now been expanded to include actions on many tissue and cell types including not only mesenchymal cells, but also epithelial cells, endothelial cells, blood cells, lymphocytes and neural cells (2, 3). With regard to functional aspects, it is well known that TGFp has multifunctional biological activities besides its conReceived January 17, 1992. Accepted for publication May 18, 1992. ‘Department of Pathology, Tokai University, School of Medicine, Kanagawa. ‘Department of Diagnostic Pathology, Tokai University Hospital, Konagawa. 3Department of Surgery, Tokai University, School of Medicine, Kanagawa. Mailing address: Koji Oda, Department of Pathology, Tokai University, School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1 1, Japan.
ventional regulatory effects on cell proliferation and differentiation (4). Among these, its effects on the synthesis of intercellular matrix proteins and the “integrin” cell receptors for them have been a focus of interest in attempts to understand the mechanisms of tumor invasion and metastasis (5-8). Several human mammary carcinoma cell lines are known t o secrete TGF p (9) and it has also been reported that TGF /3 shows growth-inhib itory effects on breast cancer cells in vitro (10-12). In human mammary tissue in vivo, on the other hand, Travers et a/. (13) have shown that T G F p messenger RNA is more abudant in carcinoma than in non-malignant breast tissue from studies of 77 biopsied breast specimens. We have also reported stronger immunohistochemical expression of T G F p in invasive breast carcinomas with lymph nodal metastasis than in those without lymph nodal metastasis (14, 15). In the present immunohistochemical study, the effects of T G F p on the induction of the extracellular matrix protein fibronectin (FN), and fibronectin receptor (FNR), one of the integrins, were examined. FN and FNR are known to play significant roles in tumor invasion and metastasis(4, 16, 17). The effects of T G F p in determining the histological types of breast carcinoma are also discussed.
MATERIALS AND METHODS Thirty -one surgically resected breast carcinomas from Japanese women were collected at the Division of Diagnostic Pathology, Tokai University Hospital. The clinical data and the specimen sources are shown in Table 1. The histological types of the breast carcinomas were determined according to the histological typing of breast tumors by the Japan Mammary Cancer Society (18). Individual cases sometimes expressed various histological types, especially with regard to the desmoplas-
646
Expression of TGFp in Human Mammary Carcinomas (Oda et a/.)
tic reaction. For this reason, among 3 1 cases of invasive ductal carcinoma, there were 12 foci showing a strong desmoplastic reaction (scirrhous lesions ; more than 50% of the tumor replaced by fibrous tissue) and 24 foci without a desmoplastic reaction (non-scirrhous lesions). All materials were frozen immediately in 0. C. T. compound and kept frozen for immunohistochemical study at -70 centigrade. Frozen sections 6 p m thick for immunohistochemistry were cut and fixed in 4% paraformaldehyde at room temperature for 30 min. The indirect immunoperoxidase method was performed using anti-TGFp1 polyclonal antibody (R & D System, Inc., U.S.A.), anti-FN monoclonal antibody (Transforming Research, Inc., U.S.A.) and anti-FNR monoclonal antibody (Telios Pharmaceuticals, Inc., U.S.A.). Sites of peroxidase reactivity were visualized using 25% diaminobenzidine, pH 7.6. The sections were counterstained with 5 % methyl green, pH 4.0. lmmunoreactivity of TGFp was divided into three grades; negative (-), positive (+) and strongly positive ( i t ) , according to the staining intensity of the
majority (more than 60%) of the cancer cells in the lesion. Fibroblasts in the non-neoplastic breast tissue stroma around the carcinoma were used as the standard for intensity of the TGFp staining. That is, in the cases with positive (+) TGFp staining, more than 60% of the examined cancer cells showed a TGF p staining intensity comparable to that of stromal fibroblasts. Strong positivity ( i t ) was defined as TGF p staining which was more intense than that of stromal fibroblasts in the majority of the cancer cells. lmmunoreactivities for FN and FNR were divided into two groups; negative (-) and positive (+), according to the imrnunoreactivity of each antigen in the majority (more than 60%) of the cancer cells.
RESULTS 1. lmmunoreactivity of TGFP, FN and FNR in 31 invasive carcinomas The results of the immunohistochemical staining of TGFp, FN and FNR are shown in Table 1. TGFp stain-
Table 1. Details of the Present Cases and lmmunohistochemistry of Mammary Carcinomas Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Patient age
51 45 41 54 46 71 52 49 40 69 50 32 47 65 60 48 53 43 33 72 43 49 43 47 44 89 44 44 49 33 68
Histology ST Sci ST MED Sci MUC Sci Sci PT ST ST PT Sci
ST ST PT PT ST
SQ MUC Sci ST Sci Sci ST Sci PT PT Sci PT ST
Maximum size (cm)
2.1 1.2 3.0 2.0 8.0 2.5 3.0 2.5 1.6 2.2 2.5 2.5 1.8 1.1 2.1 3.5 3.5 3.5 1.6 2.0 4.5 2.0
4.2 1.5 3.2 3.0 2.0 4.0 1.8 5.0 2.5
Lymph node metastasis negative negative negative positive positive positive positive positive negative negative negative positive negative negative positive positive positive negative negative negative positive positive positive positive positive positive negative negative negative positive positive
TGF ~3 S-lesion NS-lesion
/ it
/ it it
/ it it it
/ / / it
9 it
/ / / it
/
+
/ it
/ / / / /
+ +
/
+ + + it
/ it
+ / + + + + / / /
+ ++ it + + + + + + + + +
/ /
it
S-lesion
/
-
/
+ +
/
+ +
/ / / /
+ + +
FNR NS-lesion -
+ + / + -
/
+ + -
/ / /
/ /
-
+
+ + + +
/
/
+
/
/ / / / / /
-
+
/
PT : Papillo-tubular carcinoma, ST: Solid-tubular carcinoma, Sci : Scirrhous carcinoma, MUC : Mucinous carcinoma, MED : Medullary carcinoma, SQ: Squamous cell carcinoma (Japan Mammary Cancer Society).
-
+
-
-
/ /
-
Acta Pathologica Japonica 42 (9): 1992
ing was positive in all 31 cases of mammary carcinoma. TGFp was localized mainly in the cytoplasm of the cancer cells as well as in the stromal fibroblasts. The staining intensity of the carcinoma cells shown in Fig. l a was interpreted as strongly positive (+). In Fig. 2a, the staining intensity was interpreted as positive (+). Thirteen (41.9%) cases in the present study demonstrated strongly positive ( 8 )staining and 18 (58.1%) revealed positive (+) staining for TGFp. FN immunoreactivity was detected in the stroma in all 31 cases (Fig. 3b). FNR immunoreactivity was present mainly in the cancer cell membrane. The FNR staining was positive (+) in 17 (54.8%) and negative in 14 (45.2%) of the 33 carcinomas.
64 7
1 7 cases with only NS-lesions, only 2 revealed strongly positive TGFp expression. Among the 7 cases with solely S-lesions, 5 demonstrated strongly positive TGF p expression. Among the 7 cases with both S- and NSlesions, 6 demonstrated strongly positive TGF p expression in the S-lesions and 5 showed positive TGFp expression in the NS-lesions. Various degrees of TGFp expression in one case was noted in 4 cases, where the Slesion demonstrated strong TGF p expression and the NS-lesion revealed positive TGF p expression. Strongly positive TGF B expression was significantly more f r e quent in S-lesions than in NS-lesions (p
