Original Paper Oncology 1992;49:381-385
a First Department o f Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan; b Department o f Gastroenterology, Kameda General Hospital, Kamogawa, Chiba, Japan
Keywords Transforming growth factor-a Epidermal growth factor Colorectal neoplasms Adenoma-carcinoma sequence
Immunoreactive T ransforming Growth Factor-aand Epidermal Growth Factor in Colorectal Adenomas and Carcinomas
Abstract A total of 117 colorectal tissue specimens were examined immunohistochemically for the production of immunoreactive (IR-) transforming growth factor (TGF)-a and I R-epidermal growth factor (EGF). 1R-TGF-a was detect ed in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) carcinomas in situ, and 14/58 (24.1%) adenomas. 1R-EGF was detected in 14/32 (43.8%) invasive can cers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas. The staining intensity of I R-TGF-a was related to the histologic grade of malig nancy, but that of IR-EGF was not. These suggest that 1R-TGF-a plays a more important role than I R-EGF in the growth of colorectal neoplasms, and that further study of these growth factors would be helpful in understanding the biology of colorectal carcinoma.
Introduction Transforming growth factor (TGF)-a is a low mole cular weight mitogenic polypeptide that reversibly induces anchorage-independent growth of nontransformed anchorage-dependent cells and is produced by various cancer cells [1-4]. Previously we have examined several types of cancer cells for the production of immunoreactive (IR-)TGF-aand detected a large amount of I R-TGF-a in colorectal cancers [4, 5]. The endogenous TGF-a pro duced by certain cancer cells can function as an autocrine growth factor. Because the cancer cells produce IR-TGFa and express its receptor. The exogenously addition of a
monoclonal antibody against recombinant TGF-a in hibits the growth of these cancer cells in vitro [6-8]. Neoplasms of the colon and rectum can be classified histologically in order of malignant potential as invasive cancer, carcinoma in situ, and adenoma [9]. Colorectal carcinoma in situ generally can be considered as a precur sor to invasive colorectal cancer. Colorectal adenoma is regarded as a precancerous lesion. Vogelstein et al. [10] proposed a multistage carcinogenesis theory in the ad enoma-carcinoma sequence, suggesting that adenomas, which usually have only one recognizable genetic alterna tion, acquire further genetic abnormalities and undergo malignant transformation. It has been demonstrated that
Shinji Tanaka. M D First Department o f Internal Medicine Hiroshima University School of Medicine Kasumi 1-2-3, Minami-ku Hiroshima 734 (Japan)
© 1992 S. Kargcr AG, Basel 0030-2414/92/ 0495 0381 $ 2.75/0
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Shinji Tanakaa Koh-ichi Imanishib Ken Harumaa Toshitaka Tsudaa Masaharu Yoshiharaa JCo/i SumiT Goro Kajiyamaa
Materials and Methods Materials. A total of 117 colorectal tissue specimens comprised of 32 invasive cancers, 27 carcinomas in situ, and 58 adenomas were col lected at Hiroshima University Hospital from 1987 to 1989. Each specimen was obtained from a single lesion in a single patient. Inva sive cancers were obtained as surgical specimens, while carcinomas in situ and adenomas were resected by endoscopic polypectomy. The specimens were fixed immediately in 10% neutral formalin and em bedded in paraffin. The invasive cancers consisted of 12 well-differen tiated adenocarcinomas, 16 moderately differentiated adenocar cinomas, and 4 poorly differentiated adenocarcinomas. All car cinomas in situ were well-differentiated adenocarcinomas which were discovered in polypoid adenomas. In this study, the pathologic diagnosis and classification were in accord with the WHO classifica tion [9]. Antibodies. The anti-TG F-a monoclonal antibody (OALMTG01) was provided by Otsuka Assay Laboratory (Tokushima, Japan) and the anti-EG F monoclonal antibody (K.EM-10) was a kind gift from W akunaga Pharm. (Kohda, Japan). We have charac terized these antibodies previously [5]. Immunohistochemistry. Immunohistochemical staining was per formed by the avidin-biotin-peroxidase complex (ABC) method us ing an ABC Elite kit (Vector Laboratories, Burlingame, Calif.), as described previously [5]. The evaluation of the intensity o f staining was carried out by assessing the positively stained tum or cells in the
382
sections as follow; + + : over one-third were stained, + : less than one-third were stained, : negative for staining. Statistical analysis was performed by the yj test.
Results Immunohistochemical Staining. Examples of 1R-TGFaan d 1R-EGF staining are shown in figure 1 and 2. Both IR-TGF-a and IR-EGF were detected diffusely in the cytoplasm of the tumor cells. Immunohistochemical Analysis. IR-TGF-a was detect ed in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) car cinomas in situ, and 14/58 (24.1%) adenomas. IR-EGF was detected in 14/32 (43.8%) invasive cancers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas (table 1). 1R-TGF-a was detected in a significantly higher percentage of invasive cancers than was IR-EGF (P < 0 .01). The percentage of staining of 1R-TGF-a in each neoplasm differed significantly between invasive can cers and carcinomas in situ; no such difference was ob served with IR-EGF. We examined the synchronous pro duction of I R-TGF-a and 1R-EGF in the same specimens. The percentages of detectable IR-T GF-a in IR-EGF-positive specimens are shown in table 2. All (100%) 14 1REGF-positive invasive colorectal cancers also revealed the production of IR-TGF-a. IR-TGF-a was detected in 10/ 12 (83.3%) of the 1R-EGF-positive carcinomas in situ and in 7/12 (58.3%) of the IR-EGF-positive adenomas. The staining intensitiesof each lesion are shown in table 3. Invasive cancers had more intense IR-TGF-a staining
Tablel. Production of IR-TG F-a and IR-EGF Histology
Specimens, n
IR-TGF-u
IR-EG F
n
%
n
%
Invasive cancer Well. Mod. Por.
32 12 16 4
26 10 14 2
81.3a 83.3 87.5 50.0
14 7 7
43.8” 58.3 50.0
0
0.0
Carcinoma in situ
27
14
51.9e
12
44.4r
Adenoma
58
14
24. Id
12
20.7'
Well.: Well-differentiated adenocarcinoma; Mod.: moderately differentiated adenocarcinoma; Por.: poorly differentiated adeno carcinoma. a versus b: p < 0.01; a versus c, a versus d, c versus d, b versus e, f versus e: p < 0.05.
Tanaka/Imanishi/Haruma/Tsuda/ Yoshihara/Sumii/Kajiyama
Immunoreactive TGF-a and EGF in Colorectal Neoplasia
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adenomas, as well as cancers, produce IR-TGF-a, which then stimulates growth by an autocrine mechanism [11], One significant factor in the malignant potential of col orectal neoplasms is their cellular growth. Since IR-TGFa may contribute to tumor growth and its production may thus correlate with malignant potential, study of the pro duction of this growth factor in colorectal neoplasms should provide valuable insight into neoplastic transfor mation. Epidermal growth factor (EGF) is another growth fac tor that is structurally and functionally related to TGF-a and binds to a common receptor [12]. The genes for TGF-a and EGF differ in structure and chromosomal location [13], Furthermore, there are several differences in function between TGF-a and EGF'. The effects of TGF-a on an giogenesis, bone resorption, and wound healing are more potent than those of EGF [14-18]. Moreover, it has been reported that TGF-a and EGF stimulate the induction of several different growth factors, receptors, oncogenes, and enzymes [19], Recent studies have revealed that several types of cancer cells produce EGF, which accordingly might also act on the growth of cancer cells in a manner similar to TGF-a [20-22], Thus, we have examined the production of 1R-EGF in the same colorectal specimens and have compared the different patterns of expression of TGF-a and EGF.
Oncology, Vol. 49 S. Karger, Basel
Fig. 2. Immunohistochemical staining o f specimens positive for IR-EGF (middle power view), a IR-EGF staining o f a well-differen tiated colorectal adenocarcinoma. The staining pattern was alsmost the same as that of IR-TG F-a. b IR-EGF staining of a colorectal ad enoma. The staining pattern of the adenoma cells was almost the same as that o f the cancer cells.
Tanaka/Imanishi/Haruma Tsuda/Yoshihara/Sumii/ Kajiyama
Tafel I
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Fig. 1. Immunohistochemical staining of specimens positive for IR-TG F-a (middle power view), a I R-TGF-u staining of a moderate ly differentiated colorectal adenocarcinoma. IR-TG F-a was present diffusely in the cytoplasm of the cancer cells, but was not detected in the connective tissue, b I R -TG F-a staining of a colorectal adenoma. IR-TG F-a was present diffusely in the cytoplasm of the adenoma cells and was not detected in the connective tissue.
Table2. Production of IR-TG F-a in IR-EGF positive specimens Specimens, n
Histology
14 12 12
Invasive cancer Carcinoma in situ Adenoma
IR-TG F-a n
%
14 10 7
100a 83.3 58.31
er percentage. In adenomas which involved carcinoma in situ, the percentage of production of IR-TGF-a and IREGF was 22.2 and 14.8%, respectively. These figures were similar for adenomas which did not contain carcinoma in situ.
Discusssion
a versus b: p < 0.05.
Grade
Invasive cancer
Carcinoma in situ Adenoma
n
%
n
%
n
%
6
18.8
13
48.2
44
75.9
+* + + ,‘
5 21
15.6 65.6
5 9
18.5 33.3
9 5
15.5 8.6
IR-EGF -
18
56.3
15
55.6
46
79.3
+ NS + + NS
7 7
21.8 21.9
5 7
18.5 25.9
9 3
15.5 5.2
IR-TGF-a -
1
p < 0.05.
Table 4. Relationship of IR-TG F-a and IR-EG F production to adenoma size Size, mm
0-10 11 20 21-
Specimens, n
40 13 5
IR-TGF-a
IR-EG F
n
%
n
%
6 5 3
15.0 38.5 60.0
6 3 2
15.0 23.1 40.0
than carcinomas in situ or adenomas. Moreover, the IRTGF-a staining intensity of carcinomas in situ was greater than that of adenomas (p < 0.05). In contrast, there was no obvious relationship in IR-EGF staining intensity among invasive cancers, carcinomas in situ, and adenomas. For invasive cancers, the production of both IR-TGF-a and IR-EGF did not differ by histologic type or Dukes' stage. The relationships of IR-TGF-u and IR-EGF production with adenoma size are shown in tabic 4. In larger adeno mas, both I R-TGF-a and IR-EGF were detected in a high
383
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Table 3. Staining intensity for IR-TG F-a and IR-EG F
Cellular growth is one of the important factors regulat ing malignant potential of tumor. Previous studies have shown that colorectal cancer cells have a higher degree of proliferation than those of colorectal adenoma [23, 24], TGF-a and EGF both have effects on cellular growth, so examination for the production of these growth factors in colorectal neoplasms to assess their malignant potential should prove insightful. In the present study, the percentage of positivity and the intensity of IR-TGF-a staining were highest in invasive colorectal cancer, and both increased significantly with in creasing malignant potential (tables 1, 3). These findings suggest that IR-TGF-a is useful in understanding the ma lignant potential of each colorectal neoplasm in the ad enoma-carcinoma sequence. It has been reported that ex pression of the EGF receptor is detected in almost all col orectal cancers [25, 26], while another study indicated EGF receptor expression not only in colorectal cancers but also in colorectal adenomas [11,27], Increased expres sion of both TGF'-a and EGF receptor mRNA has been shown in several types of cancer cells [28], Our results, al ong with these reports, reveal that IR-TGF-a plays an im portant role in the growth of colorectal neoplasms, due perhaps to interactions of TGF-a with the EGF receptor. In several types of cancers, previous reports have in dicated that the production of IR-EGF correlates to the aggressiveness of malignancy [20, 21]. However, in this study of colorectal neoplasms, IR-TGF-a seemed to cor relate better than IR-EGF. The reasons were as follows: (1) Although IR-EGF-positive lesions followed the same order of percentage as those positive for I R-TGF-u. the percentage of IR-F.GF positivity in invasive cancer was almost the same as that of carcinoma in situ. Furthermore, the percentage of I R-TGF-a production in invasive cancer was significantly higher than that of 1R-EGF (table 1). (2) The I R-TGF-u staining intensity was greater in invasive cancer than in carcinoma in situ, while the IR-EGF stain ing intensity was similar in both types of cancers (table 3). (3) Almost all IR-EG F-posili ve invasive cancer specimens and carcinoma in situ specimens were also IR-TGF-u po sitive (table 2).
This study revealed that I R-TGF-a production in inva sive cancers has no relationship to the Dukes' stage. Inva sive and metastatic potentials may depend upon factors other than the production of 1R-TGF-a. For example, it has been reported that platelet-derived growth factor (PDGF) plays an important role in the establishment of metastasis of colorectal cancer cells [29], Extracellular degenerative enzymes are assumed to play important roles in tumor invasion and metastasis [30, 31]. Recently it has been shown that TGF-a and EGF stimulation causes the differential induction of several growth factors, their receptors and the enzymes [19], It will be important to ex amine the mechanisms of the invasion and metastasis in colorectal cancer, including the relationship with TGF-a and EGF. The size of colorectal adenomas was related not only to the percentage of I R-TGF-a staining positivity, but also to thatoflR -EG F (table 4), suggesting that adenoma grow th might be related to the production of these growth factors. In the case of adenomas, the percentages of IR-TGF-a positivity and 1R-EGF positivity were similar for those which involved carcinoma in situ and those which did not. This may reflect the fact that the adenoma tissue which in volves carcinoma in situ differs from the focal carcinoma tissue in biologic characteristics as well as in pathologic features.
In summary, the growth of colorectal neoplasms may involve the actions of both TGF-a and EGF, with TGF-a playing a more important role than EGF. These results may be useful in better understanding the sequence of events in colorectal carcinogenesis. In the growth of col orectal neoplasms, the significance of these two growth factors with linked biochemical pathways, relative to the role of other growth factors or growth factor-independent mechanisms, is not known at present. Recent progress has demonstrated the evidence of several substances homolo gous to the EGF family proteins (amphiregulin, etc.) [32], The expression of several growth factors and their recep tors has been reported in colorectal neoplasms as well as in other neoplasms. Further studies are needed to elucidate whether these growth factors and thei r receptors expressed by colorectal neoplasms are actually functional.
Acknowledgements We are grateful to MissChisa Miki for her excellent technical as sistance. The gifts of OAL-MTGOl from Otsuka Assay Laboratory (Tokushima, Japan) and KEM-10from Wakunaga Pharm. (Kohda, Japan) are also gratefully acknowledged. This work was supported in part by theTsuchiya Foundation.
References
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Immunoreactive T G F-a and EG F in Colorectal Neoplasia
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15 Ibbotson KJ. Twardzik DR. D’Souza SM. Hargreaves WR, Todaro G J. Mundy G R: Stimulation of bone resorption in vitro by transforming growth factor-alpha. Science 1985;228:1007-1009. 16 Ibbotson KJ, Harrold J. Gowcn M. D'Souza S, Smith DD. Winkler ME. Derynk R. Mundy GR: Human recombinant transforming growth factor a stimulates bone resorption and inhibits formation in vitro. Proc Natl Acad Sei USA 1986;83:2228-2232. 17 Coffey RJ. Derynk R. Wilcox JN. Bringman TS. Goustin AS, Moses HL. Pittelkow M R: Production and auto-induction of transform ing growth factor-« in human kératinocytes. Nature 1987;328:817-820. 18 GottliebAB.ChangCK.PosnettDN.Fanelli B. Tam JP: Detection of transforming growth factor a in normal, malignant, and hyperproliferative human kératinocytes. J Exp Med 1988;167:670-675. 19 Yoshida K. Tsujino T. Yasui W. Kameda T, SanoT. Nakayama H. TogeT, Tahara E: In duction of growth factor-receptor and metalloproteinase genes by epidermal growth factor and or transforming growth factor-a in human gastric carcinoma cell line MKN-28. Jpn J Cancer Res 1990:81:793-798. 20 Tahara E. Sumiyoshi H. HataJ. Yasui W. Taniyama K, Hayashi T. Nagae S. Sakamoto S: Human epidermal growth factor in gastric car cinoma as a biological marker of high malig nancy. Jpn J Cancer Res 1986;77:145-152.
a First Department o f Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan; b Department o f Gastroenterology, Kameda General Hospital, Kamogawa, Chiba, Japan
Keywords Transforming growth factor-a Epidermal growth factor Colorectal neoplasms Adenoma-carcinoma sequence
Immunoreactive T ransforming Growth Factor-aand Epidermal Growth Factor in Colorectal Adenomas and Carcinomas
Abstract A total of 117 colorectal tissue specimens were examined immunohistochemically for the production of immunoreactive (IR-) transforming growth factor (TGF)-a and I R-epidermal growth factor (EGF). 1R-TGF-a was detect ed in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) carcinomas in situ, and 14/58 (24.1%) adenomas. 1R-EGF was detected in 14/32 (43.8%) invasive can cers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas. The staining intensity of I R-TGF-a was related to the histologic grade of malig nancy, but that of IR-EGF was not. These suggest that 1R-TGF-a plays a more important role than I R-EGF in the growth of colorectal neoplasms, and that further study of these growth factors would be helpful in understanding the biology of colorectal carcinoma.
Introduction Transforming growth factor (TGF)-a is a low mole cular weight mitogenic polypeptide that reversibly induces anchorage-independent growth of nontransformed anchorage-dependent cells and is produced by various cancer cells [1-4]. Previously we have examined several types of cancer cells for the production of immunoreactive (IR-)TGF-aand detected a large amount of I R-TGF-a in colorectal cancers [4, 5]. The endogenous TGF-a pro duced by certain cancer cells can function as an autocrine growth factor. Because the cancer cells produce IR-TGFa and express its receptor. The exogenously addition of a
monoclonal antibody against recombinant TGF-a in hibits the growth of these cancer cells in vitro [6-8]. Neoplasms of the colon and rectum can be classified histologically in order of malignant potential as invasive cancer, carcinoma in situ, and adenoma [9]. Colorectal carcinoma in situ generally can be considered as a precur sor to invasive colorectal cancer. Colorectal adenoma is regarded as a precancerous lesion. Vogelstein et al. [10] proposed a multistage carcinogenesis theory in the ad enoma-carcinoma sequence, suggesting that adenomas, which usually have only one recognizable genetic alterna tion, acquire further genetic abnormalities and undergo malignant transformation. It has been demonstrated that
Shinji Tanaka. M D First Department o f Internal Medicine Hiroshima University School of Medicine Kasumi 1-2-3, Minami-ku Hiroshima 734 (Japan)
© 1992 S. Kargcr AG, Basel 0030-2414/92/ 0495 0381 $ 2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/14/2019 10:47:23 PM
Shinji Tanakaa Koh-ichi Imanishib Ken Harumaa Toshitaka Tsudaa Masaharu Yoshiharaa JCo/i SumiT Goro Kajiyamaa
Materials and Methods Materials. A total of 117 colorectal tissue specimens comprised of 32 invasive cancers, 27 carcinomas in situ, and 58 adenomas were col lected at Hiroshima University Hospital from 1987 to 1989. Each specimen was obtained from a single lesion in a single patient. Inva sive cancers were obtained as surgical specimens, while carcinomas in situ and adenomas were resected by endoscopic polypectomy. The specimens were fixed immediately in 10% neutral formalin and em bedded in paraffin. The invasive cancers consisted of 12 well-differen tiated adenocarcinomas, 16 moderately differentiated adenocar cinomas, and 4 poorly differentiated adenocarcinomas. All car cinomas in situ were well-differentiated adenocarcinomas which were discovered in polypoid adenomas. In this study, the pathologic diagnosis and classification were in accord with the WHO classifica tion [9]. Antibodies. The anti-TG F-a monoclonal antibody (OALMTG01) was provided by Otsuka Assay Laboratory (Tokushima, Japan) and the anti-EG F monoclonal antibody (K.EM-10) was a kind gift from W akunaga Pharm. (Kohda, Japan). We have charac terized these antibodies previously [5]. Immunohistochemistry. Immunohistochemical staining was per formed by the avidin-biotin-peroxidase complex (ABC) method us ing an ABC Elite kit (Vector Laboratories, Burlingame, Calif.), as described previously [5]. The evaluation of the intensity o f staining was carried out by assessing the positively stained tum or cells in the
382
sections as follow; + + : over one-third were stained, + : less than one-third were stained, : negative for staining. Statistical analysis was performed by the yj test.
Results Immunohistochemical Staining. Examples of 1R-TGFaan d 1R-EGF staining are shown in figure 1 and 2. Both IR-TGF-a and IR-EGF were detected diffusely in the cytoplasm of the tumor cells. Immunohistochemical Analysis. IR-TGF-a was detect ed in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) car cinomas in situ, and 14/58 (24.1%) adenomas. IR-EGF was detected in 14/32 (43.8%) invasive cancers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas (table 1). 1R-TGF-a was detected in a significantly higher percentage of invasive cancers than was IR-EGF (P < 0 .01). The percentage of staining of 1R-TGF-a in each neoplasm differed significantly between invasive can cers and carcinomas in situ; no such difference was ob served with IR-EGF. We examined the synchronous pro duction of I R-TGF-a and 1R-EGF in the same specimens. The percentages of detectable IR-T GF-a in IR-EGF-positive specimens are shown in table 2. All (100%) 14 1REGF-positive invasive colorectal cancers also revealed the production of IR-TGF-a. IR-TGF-a was detected in 10/ 12 (83.3%) of the 1R-EGF-positive carcinomas in situ and in 7/12 (58.3%) of the IR-EGF-positive adenomas. The staining intensitiesof each lesion are shown in table 3. Invasive cancers had more intense IR-TGF-a staining
Tablel. Production of IR-TG F-a and IR-EGF Histology
Specimens, n
IR-TGF-u
IR-EG F
n
%
n
%
Invasive cancer Well. Mod. Por.
32 12 16 4
26 10 14 2
81.3a 83.3 87.5 50.0
14 7 7
43.8” 58.3 50.0
0
0.0
Carcinoma in situ
27
14
51.9e
12
44.4r
Adenoma
58
14
24. Id
12
20.7'
Well.: Well-differentiated adenocarcinoma; Mod.: moderately differentiated adenocarcinoma; Por.: poorly differentiated adeno carcinoma. a versus b: p < 0.01; a versus c, a versus d, c versus d, b versus e, f versus e: p < 0.05.
Tanaka/Imanishi/Haruma/Tsuda/ Yoshihara/Sumii/Kajiyama
Immunoreactive TGF-a and EGF in Colorectal Neoplasia
Downloaded by: Nagoya University 133.6.82.173 - 1/14/2019 10:47:23 PM
adenomas, as well as cancers, produce IR-TGF-a, which then stimulates growth by an autocrine mechanism [11], One significant factor in the malignant potential of col orectal neoplasms is their cellular growth. Since IR-TGFa may contribute to tumor growth and its production may thus correlate with malignant potential, study of the pro duction of this growth factor in colorectal neoplasms should provide valuable insight into neoplastic transfor mation. Epidermal growth factor (EGF) is another growth fac tor that is structurally and functionally related to TGF-a and binds to a common receptor [12]. The genes for TGF-a and EGF differ in structure and chromosomal location [13], Furthermore, there are several differences in function between TGF-a and EGF'. The effects of TGF-a on an giogenesis, bone resorption, and wound healing are more potent than those of EGF [14-18]. Moreover, it has been reported that TGF-a and EGF stimulate the induction of several different growth factors, receptors, oncogenes, and enzymes [19], Recent studies have revealed that several types of cancer cells produce EGF, which accordingly might also act on the growth of cancer cells in a manner similar to TGF-a [20-22], Thus, we have examined the production of 1R-EGF in the same colorectal specimens and have compared the different patterns of expression of TGF-a and EGF.
Oncology, Vol. 49 S. Karger, Basel
Fig. 2. Immunohistochemical staining o f specimens positive for IR-EGF (middle power view), a IR-EGF staining o f a well-differen tiated colorectal adenocarcinoma. The staining pattern was alsmost the same as that of IR-TG F-a. b IR-EGF staining of a colorectal ad enoma. The staining pattern of the adenoma cells was almost the same as that o f the cancer cells.
Tanaka/Imanishi/Haruma Tsuda/Yoshihara/Sumii/ Kajiyama
Tafel I
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Fig. 1. Immunohistochemical staining of specimens positive for IR-TG F-a (middle power view), a I R-TGF-u staining of a moderate ly differentiated colorectal adenocarcinoma. IR-TG F-a was present diffusely in the cytoplasm of the cancer cells, but was not detected in the connective tissue, b I R -TG F-a staining of a colorectal adenoma. IR-TG F-a was present diffusely in the cytoplasm of the adenoma cells and was not detected in the connective tissue.
Table2. Production of IR-TG F-a in IR-EGF positive specimens Specimens, n
Histology
14 12 12
Invasive cancer Carcinoma in situ Adenoma
IR-TG F-a n
%
14 10 7
100a 83.3 58.31
er percentage. In adenomas which involved carcinoma in situ, the percentage of production of IR-TGF-a and IREGF was 22.2 and 14.8%, respectively. These figures were similar for adenomas which did not contain carcinoma in situ.
Discusssion
a versus b: p < 0.05.
Grade
Invasive cancer
Carcinoma in situ Adenoma
n
%
n
%
n
%
6
18.8
13
48.2
44
75.9
+* + + ,‘
5 21
15.6 65.6
5 9
18.5 33.3
9 5
15.5 8.6
IR-EGF -
18
56.3
15
55.6
46
79.3
+ NS + + NS
7 7
21.8 21.9
5 7
18.5 25.9
9 3
15.5 5.2
IR-TGF-a -
1
p < 0.05.
Table 4. Relationship of IR-TG F-a and IR-EG F production to adenoma size Size, mm
0-10 11 20 21-
Specimens, n
40 13 5
IR-TGF-a
IR-EG F
n
%
n
%
6 5 3
15.0 38.5 60.0
6 3 2
15.0 23.1 40.0
than carcinomas in situ or adenomas. Moreover, the IRTGF-a staining intensity of carcinomas in situ was greater than that of adenomas (p < 0.05). In contrast, there was no obvious relationship in IR-EGF staining intensity among invasive cancers, carcinomas in situ, and adenomas. For invasive cancers, the production of both IR-TGF-a and IR-EGF did not differ by histologic type or Dukes' stage. The relationships of IR-TGF-u and IR-EGF production with adenoma size are shown in tabic 4. In larger adeno mas, both I R-TGF-a and IR-EGF were detected in a high
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Table 3. Staining intensity for IR-TG F-a and IR-EG F
Cellular growth is one of the important factors regulat ing malignant potential of tumor. Previous studies have shown that colorectal cancer cells have a higher degree of proliferation than those of colorectal adenoma [23, 24], TGF-a and EGF both have effects on cellular growth, so examination for the production of these growth factors in colorectal neoplasms to assess their malignant potential should prove insightful. In the present study, the percentage of positivity and the intensity of IR-TGF-a staining were highest in invasive colorectal cancer, and both increased significantly with in creasing malignant potential (tables 1, 3). These findings suggest that IR-TGF-a is useful in understanding the ma lignant potential of each colorectal neoplasm in the ad enoma-carcinoma sequence. It has been reported that ex pression of the EGF receptor is detected in almost all col orectal cancers [25, 26], while another study indicated EGF receptor expression not only in colorectal cancers but also in colorectal adenomas [11,27], Increased expres sion of both TGF'-a and EGF receptor mRNA has been shown in several types of cancer cells [28], Our results, al ong with these reports, reveal that IR-TGF-a plays an im portant role in the growth of colorectal neoplasms, due perhaps to interactions of TGF-a with the EGF receptor. In several types of cancers, previous reports have in dicated that the production of IR-EGF correlates to the aggressiveness of malignancy [20, 21]. However, in this study of colorectal neoplasms, IR-TGF-a seemed to cor relate better than IR-EGF. The reasons were as follows: (1) Although IR-EGF-positive lesions followed the same order of percentage as those positive for I R-TGF-u. the percentage of IR-F.GF positivity in invasive cancer was almost the same as that of carcinoma in situ. Furthermore, the percentage of I R-TGF-a production in invasive cancer was significantly higher than that of 1R-EGF (table 1). (2) The I R-TGF-u staining intensity was greater in invasive cancer than in carcinoma in situ, while the IR-EGF stain ing intensity was similar in both types of cancers (table 3). (3) Almost all IR-EG F-posili ve invasive cancer specimens and carcinoma in situ specimens were also IR-TGF-u po sitive (table 2).
This study revealed that I R-TGF-a production in inva sive cancers has no relationship to the Dukes' stage. Inva sive and metastatic potentials may depend upon factors other than the production of 1R-TGF-a. For example, it has been reported that platelet-derived growth factor (PDGF) plays an important role in the establishment of metastasis of colorectal cancer cells [29], Extracellular degenerative enzymes are assumed to play important roles in tumor invasion and metastasis [30, 31]. Recently it has been shown that TGF-a and EGF stimulation causes the differential induction of several growth factors, their receptors and the enzymes [19], It will be important to ex amine the mechanisms of the invasion and metastasis in colorectal cancer, including the relationship with TGF-a and EGF. The size of colorectal adenomas was related not only to the percentage of I R-TGF-a staining positivity, but also to thatoflR -EG F (table 4), suggesting that adenoma grow th might be related to the production of these growth factors. In the case of adenomas, the percentages of IR-TGF-a positivity and 1R-EGF positivity were similar for those which involved carcinoma in situ and those which did not. This may reflect the fact that the adenoma tissue which in volves carcinoma in situ differs from the focal carcinoma tissue in biologic characteristics as well as in pathologic features.
In summary, the growth of colorectal neoplasms may involve the actions of both TGF-a and EGF, with TGF-a playing a more important role than EGF. These results may be useful in better understanding the sequence of events in colorectal carcinogenesis. In the growth of col orectal neoplasms, the significance of these two growth factors with linked biochemical pathways, relative to the role of other growth factors or growth factor-independent mechanisms, is not known at present. Recent progress has demonstrated the evidence of several substances homolo gous to the EGF family proteins (amphiregulin, etc.) [32], The expression of several growth factors and their recep tors has been reported in colorectal neoplasms as well as in other neoplasms. Further studies are needed to elucidate whether these growth factors and thei r receptors expressed by colorectal neoplasms are actually functional.
Acknowledgements We are grateful to MissChisa Miki for her excellent technical as sistance. The gifts of OAL-MTGOl from Otsuka Assay Laboratory (Tokushima, Japan) and KEM-10from Wakunaga Pharm. (Kohda, Japan) are also gratefully acknowledged. This work was supported in part by theTsuchiya Foundation.
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