Clinical Significance of ras p21 Overexpression for Patients with an Advanced Colorectal Cancer Masaki Miyahara, M.D., Takao Saito, M.D., Kazutoshi Kaketani, M.D., Koichi Sato, M.D., Akihiko Kuwahara, M.D., Katsuhiro Shimoda, M.D., Michio Kobayashi, M.D. From the Department of Surgery I, Medical College of Oita, Oita, Japan The expression of ras oncogene product p21 was examined in 45 paraffin-embedded sections of primary advanced colorectal cancers, using the anti-v-H-ras p21 monoclonal antibody Y13-259. Fourteen of these specimens (31 percent) were stained positively. The incidence of lymphatic vessel invasion of cancer cells and lymph node metastasis correlated statistically with the overexpression of ras p21. The depth of invasion and incidence of liver metastasis in the p21-positive group were more prominent than in the p21-negative group. Statistically significant differences were evident in operative curability and clinical stage at initial surgery and in the longterm survival rate between these groups (P < 0.05). We propose that ras p21 overexpression may serve as a marker to predict the prognosis of colorectal cancer. [Key words: Colorectal cancer; ras p21 overexpression; Immunohistochemical staining; Clinicopathologic findings; Malignant potential; Prognosis] Miyahara M, Saito T, Kaketani K, Sato K, Kuwahara A, Shimoda K, Kobayashi M. Clinical significance of ras p21 overexpression for patients with an advanced colorectal cancer. Dis Colon Rectum 1991 ;34:1097-1102. he p r e s e n c e or absence of metastasis, 1' 2 d e p t h of tumor invasion, and venous and lymphatic vessel p e r m e a t i o n provide prognostic information in patients with colorectal cancer. 3'4 Most of these histopathologic findings are made postsurgically. It seems important to evaluate the potential malignancy of the cancer in each patient preoperatively. Among the o n c o g e n e family, ras e n c o d e s 21,000dalton guanosine triphosphate-binding proteins, p21, 5 and is involved in signal transduction from the m e m b r a n e r e c e p t o r to the cell interior. 6 The overexpression of ras p21 seems to relate to transformation, tumorigenicity, and malignancy in certain species of cancer cells in experimental animals. 7-.1 In humans, the increased expression of ras p21 was n o t e d to be associated with aggressiveness of tumors in the colorectum, .2 breast, .3' ,4 and prostate.15
T
Address reprint requests to Dr. Miyahara:Department of Surgery I, Medical College of Oita, 1-1 Idaigaoka Hasamamachi, Oitagun, Oita 879-56,Japan. 1097
The object of the present study was to search for the overexpression of the ras o n c o g e n e in primary colorectal cancer.
MATERIALS A N D M E T H O D S Patients I n c l u d e d in this study were 45 Japanese patients with colorectal cancer (16 colon and 29 rectal) treated in our clinic. The m e a n age of the patients was 60.5 years (range, 33-84 years). Twenty-nine patients were men, and 16 were w o m e n . All patients u n d e r w e n t resection of the primary lesion with l y m p h a d e n e c t o m y (25 colectomy, 20 rectal amputation). Thirty-one of these patients underwent curative resection for primary colorectal cancers at the time of the initial surgery, whereas the remaining 14 (10 with liver metastasis, 3 with distant lymph n o d e metastasis, and 1 with peritoneal dissemination) u n d e r w e n t noncurative resection. H e p a t e c t o m y was p e r f o r m e d in five patients with a liver metastasis, and hepatic arterial cannulation was d o n e for the remaining five patients. E x t e n d e d l y m p h a d e n e c t o m y was p e r f o r m e d in three patients with distant lymph n o d e metastasis. Cisplatin was given intraperitoneally to o n e patient with peritoneal dissemination of the cancer. 5-Fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-FU (tegafur) or uracil/FT was prescribed orally for patients treated by curative resection. A combination c h e m o t h e r a p y of mitomycin C, Adriamycin (Farmitalia, Milan, Italy), and 5-FU was given to patients for w h o m noncurative resection was done. Clinicopathologic findings and evaluations of operative p r o c e d u r e s w e r e according to "General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus" established by the Japanese Research Society for Cancer of Colon TM
1098
MIYAHARA E T AL
and Rectum, ~6 and clinical staging was according to the Union Internationale Contre le Cancer TNM classification.
Dis Colon Rectum, December 1991
containing 0.01 percent H202 for 5 minutes, washed, and counterstained with hematoxylin.
Statistics Anti-ras p 2 1 M o n o c l o n a l A n t i b o d y Biotinylated anti-v-H-ras p21 antibody (Y13-259) was purchased from Oncogene Science, Inc. (Mineola, NY). This is a rat monoclonal antibody directed against the p21 ras gene translational product of the Harvey strain of rat sarcoma virus 17 and reacts with the p21 translational products of H-, K-, and N-c-ras human oncogenes. 18' 19
Immunohistochemical Staining of Tissues The avidin-biotin-complex immunoperoxidase assay method 2~ was used to stain the excised tissues. Four-micron sections were deparaffinized and immersed in methanol containing 0.3 percent H202 for 30 minutes to eliminate the endogenous peroxidase activity. The sections were then washed with phosphate-buffered saline (PBS), normal mouse serum was added, and the preparations were incubated for 20 minutes at room temperature. The slides were then incubated at 4~ for eight hours with biotinylated anti-v-H-ras p21 antibody (2 #g/ml), washed with PBS, and incubated at room temperature for 15 minutes after the addition of peroxidase-avidin complexes. After washing with PBS, the slides were treated with 0.05 percent diaminobenzidine in 0.05 M Tris-HC1 (pH 7.2)
Correlations between p21 overexpression and pathologic features were examined using the chisquared test or Fisher's exact test. Survival rate was estimated on the basis of the Kaplan-Meier method, and the generalized Wilcoxon test was used for analysis. Pvalues of less than 0.05 were considered to be statistically significant. RESULTS
Expression of p21 in Colorectal Cancer Tissues Two micrograms per milliliter of Y13-259 were used since the cytoplasm of the cancer cells was well stained, but the adjacent normal mucosa was less stained with this concentration (Fig. 1). The positivity of the staining was classified into two groups, based on the ratio of ras p21-positive cancer ceils; specimens containing over 50 percent of ras p21-positive cancer cells were classed as the positive group and those under 50 percent as the negative group. The intensity of the staining varied with each specimen. As shown in Table 1, 45 patients were divided into two groups: the positive group (14 patients; 31 percent) and the negative group (31 patients; 69 percent). Backgrounds of
Figure 1. Immunohistochemical staining of ras p21 with monoclonal antibody Y13-259 and the avidin-biotin-peroxidase complex technique. A. Positive case: peroxidase reaction is seen in the cytoplasm of cancer cells. B. Negative case. (Hematoxylin counterstain, x400.)
ras p21 EXPRESSION IN COLORECTAL CANCER
Vol. 34, No. 12
Table 1. ras p21 Overexpression in Colorectal Cancer ras p21 Overexpression No. of patients (%)
Positive
Negative
14 (31)
31 (69)
Age (years) Range Mean Sex Male (%) Female (%) Duration of symptoms __7 months (%)
Negative (n = 31)
P Value
42-84 33-79 63.6_+11.7 59.1_+11.8 0.113 9 (64) 5 (36) 3 (21) 3 (21) 4 (29) 4 (29)
ras p21 Overexpression
45 (100)
ras p21 Overexpression Positive (n = 14)
Table 3. ras p21 Overexpression and Tumor Size
Total
Table 2. ras p21 Overexpression and Clinical Characteristics
20 (65) 11 (35) 5 (16) 10 (32) 8 (26) 8 (26)
1099
Length (cm)* Widtht 0-30 (%) 31-60 (%) 61-100 (%)
Positive (n = 14)
Negative (n = 31)
5.5 + 1.4
4.6 _+ 1.9
0 (0) 2 (14) 9 (86)
5 (17) 7 (23) 19 (61)
P Value
0.0491"
0.189
* Mean longitudinal length of the tumor. t Significant difference. :[: Ratio of tumor-occupying width to entire circumference of intestinal wall. Table 4. ras p21 Overexpression and Histopathologic Findings
1.000
0.897
the patients were compared (Table 2). Differences between the two groups with regard to sex, mean age, and duration of symptoms before admission were not significant. Expression of p2 1 and Clinicopathologic Findings Correlations of the overexpression of ras p21 and clinicopathologic findings were also investigated. The longitudinal length of the tumor was significantly greater in the positive than in the negative group (Table 3). However, there was no significant difference between the two groups with regard to the ratio of tumor-occupying width to entire circumference of the intestinal wall. Histologic types were not related to the overexpression of ras p21 (Table 4). From the 14 patients of the ras p21-positive group, there was no specimen in which carcinoma was limitedto within the proper muscle layer (pro). In four specimens (29 percent) of this group, the malignant cells extended beyond the proper muscle layer but were not exposed to the serosal surface (ss) or the adventitial layer (al). Infiltration to the serosal or adventitial layer (s,a2) or to the neighboring tissue (s~,a,) was obvious in the other 10 specimens (71
ras p21 Overexpression
Histologic type* Well (%) Moderately (%) Poorly (%) Mucinous (%) Depth of invasionipm (%) ss,a~ (%) s,a2; s~,a,(%) Lymphatic invasion (+):l: (%) Venous invasion (+) (%)
P Value
Positive (n = 14)
Negative (n = 31)
10 (71) 3 (21) 1 (7) 0 (0)
23 (74) 6 (19) 0 (0) 2 (6)
0.369
0 (0) 24 (29) 10 (71) 14 (100)
4 (13) 16 (52) 11 (35) 18 (58)
0.060 0.005w
6 (43)
5 (16)
0.124
* Well: well-differentiated adenocarcinoma; Moderately: moderately differentiated adenocarcinoma; Poorly: poorly differentiated adenocarcinoma; Mucinous: mucinous carcinoma. 1 pm: cancer is confined to within proper muscular layer; ss,al: cancer invades serosa or adventitia to some extent; s,a2: cancer invades serosa or adventitia extensively; s~,ag cancer infiltrates neighboring tissue. ~:(+): Present. w Significant difference. percent). On the other hand, in the ras p21-negative group, there were only 11 of 31 specimens (35 percent) with invasion of s,a2 or si,ai (Table 4). Lymphatic vessel invasion of cancer cells was detected in all 14 specimens in the ras p21-positive group and in 18 of 31 (58 percent) in the negative group, with a statistically significant difference (P < 0.05). Venous invasion of cancer cells was present in 6 of 14 specimens (43 percent) in the ras
1100
MIYAHARA E T AL
p21-positive group and in 5 of 31 (16 percent) in the negative group. Venous invasion tended to be more frequent in specimens with ras p21 overexpression than in those without it, but with no statistical significance. Twenty-three patients had lymph node metastases and ten liver metasatases at the time of the initial surgery. There was a positive lymph node metastasis in 79 percent of those in the ras p21positive group but in only 39 percent of those in the negative group (Table 5). The incidence of liver metastasis in the ras p21-positive group was also higher than in the negative group. Clinical stages and operative curabilities in patients with or without overexpression of p21 are shown in Table 6. There was a significant difference with regard to TNM stage between the two groups. The rates of noncurative resection in the p21-positive group and in the p21-negative group were 64 percent and 16 percent, respectively. Table 5. ras p21 Overexpression and Metastasis ras p21 Overexpression
Lymph node metastasis Positive (%) Negative (%) Liver metastasis Positive (%) Negative (%)
P Value
Positive (n=14)
Negative (n=31)
11 (79) 3 (21)
12 (39) 19 (61)
0.029*
6 (43) 8 (57)
4 (13) 27 (87)
0.069
* Significant difference. Table 6. ras p21 Overexpression and Clinical Stage and Curability of Operation ras p21 Overexpression
Clinical stage* la, Ib, II (%) III, IV (%) Curability of operation:l: Curative (%) Noncurative (%)
P Value
Positive (n=14)
Negative (n=31)
2 (14) 12 (86)
17 (55) 14 (45)
0.022t
5 (36) 9 (64)
26 (84) 5 (16)
0.0041"
* TNM stage. 1" Significant difference. 1: Evaluation of operative procedure according to "General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus". 16
100
Dis Colon Rectum, December 1991
-•6-6--8-J----•
....... 6- rasop21 negative (n=31) .......
.._J
~
1 ........
i
1,
..... , ..... ,, 6 --L--u--t---t 59.7
~
S0
J
i
J
i
ras p21 positive (n = 14)
-
~-
0
I
1
I
I
i I
2 3 4 YEARSAFTEROPERATION
16.1 I
5
Figure 2. Survival curves of patients with resected colorectal cancer, with or without overexpression of ras p21. The difference was statistically significant (P < 0.05). Figure 2 shows overall survival curves of the groups, with or without the overexpression of ras p21. The five-year survival rates of the p21-positive and negative groups were 16.1 percent and 59.7 percent, respectively; the difference was statistically significant. DISCUSSION The ras oncogene family was seen to relate with tumor development, progression, and metastasis, either following activation of the gene resulting from a point mutation at amino acids 12 or 6119' 21 or following overexpression of the gene product, p21.12 15,22 Monoclonal antibodies to ras p21 can facilitate detection of the expressed ras p21 in human tissues. The antibody Y13-259 used in the present study and the RAP-512 are widely used to recognize the p21 product of human ras genes since there is a specific reaction with ras p21 in fixed tissues. 12' 17-19,23 Czerniak e t al. 23 noted that the intensity of staining and the proportion of positively reacting cells were similar with Y13-259 and RAP-5 in cases of gastric cancer. With regard to human colorectal cancer, there are conflicting reports on the relationship between ras p21 overexpression and tumor progression. 12'24-26 Immunohistochemical studies done using RAP antibodies showed that the increased ras p21 expression correlates with the depth of invasion of colonic carcinomas. 12' 24 On the other hand, Gallick e t al. 25 reported that relatively early Dukes' stages of tumors also have elevated levels of ras p21.
Vol. 34, No. 12
ras p21 EXPRESSION IN COLORECTAL CANCER
We observed that overexpression of ras p21 statistically related to clinicopathologic factors is considered to be closely linked to a p o o r prognosis, i . e . , size of tumor, lymphatic vessel invasion, and liver metastasis. T h e r e was a t e n d e n c y toward a relation, albeit not statistically, to the d e p t h of invasion, venous invasion, and liver metastasis. T h e r e was also a significant difference b e t w e e n the ras p21-positive group and negative group concerning long-term survival. Our present study clearly s h o w e d that the prognosis of patients with ras p21 overexpression was p o o r e r than that of those without it. The lower survival rate in the p21-positive patients was based on the finding that patients in the positive group had a higher rate of noncurative resection at the initial surgery. Factors evaluated as noncurative were liver metastasis, distant lymph n o d e metastasis, or peritoneal dissemination. Thus, colorectal cancer highly expressing ras p21 has a p o t e n t biologic malignancy potential, as reflected by tumor progression, metastasis, and a p o o r prognosis. Various clinicopathologic prognostic factors include histologic type, d e p t h of invasion, and vessel involvement. 1-4 We f o u n d that overexpression of ras p21 c o r r e s p o n d e d with the pathologic progression and clinical stage of the cancer. Therefore, i m m u n o h i s t o c h e m i c a l d e t e c t i o n of overexpression with Y13-259, especially in preoperatively b i o p s i e d tissues, should b e c o n s i d e r e d for evaluation of the a d v a n c e m e n t of the cancer. O n c e this e v i d e n c e has b e e n obtained, suitable treatment can be prescribed. CONCLUSION Detection of ras p21 overexpression in colorectal cancer by the avidin-biotin-complex i m m u n o p e r oxidase assay m e t h o d can serve as a useful tool for clinical evaluation of the potential malignancy of tumors in patients. ACKNOWLEDGMENT The authors thank M. Ohara for critical comments.
REFERENCES 1. olson RM, Perencevich NP, Malcolm AW, Chaffey JT, Wilson RE. Pattern of recurrence following cu-
1101
rative resection adenocarcinoma of the colon and rectum. Cancer 1980;45:2969-74. 2. Eisenberg B, Decosse JI, Hartford F, Michalek J. Carcinoma of the colon and rectum: the natural history reviewed in 1704 patients. Cancer 1982; 49:1131-4. 3. Muraoka Y, Maetani S, Tobe T. Statistical analysis of pathological factors influencing prognosis of colorectal carcinoma. J Jpn Surg Soc 1988;89:181-91. 4. Kotanagi H, Nagasawa O, Niwa M, Takahashi M, Narisawa T, Koyama K. Prediction of the hematogenic metastasis and local recurrence of rectal cancer by quantification of the clinico-pathological factors. J Jpn surg Soc 1988;89:1022-7. 5. Papageorge A, Lowy DR, Scolnick EM. Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol 1982;44:509-19. 6. Hurley JB, Simon MI, Teplow DB. Homologies between signal transducing G proteins and gene products. Science 1984;226:860-2. 7. Thorgeirsson UP, Turpeenniemi-Hujanen T, WilliamsJE, et al. NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol 1985;5:259-62. 8. Bernstein SC, Winberg RA. Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA. Proc Natl Acad Sci USA 1985;82:1726-30. 9. Spandidos DA, Wilkie NM. Malignant transformation of early passage rodent cells by a single mutated human oncogene. Nature 1984;310:469-75. 10. Muschel RJ, WilliamsJE, Lowy DR, Liotta LA. Harvey ras induction of metastatic potential depends upon oncogene activation and the type of recipient cell. Am J Pathol 1985; 121:1-8. 11. Bradley MO, Kraynak AR, Storer RD, Gibbs JB. Experimental metastasis in nude mice of NIH3T3 cells containing various ras genes. Proc Natl Acad Sci USA 1986;83:5277-81. 12. Thor A, Hand PH, Wunderlich D, Caruso A, Muraro R, Schlom J. Monoclonal antibodies define differential ras gene expression in malignant and benign colonic disease. Nature 1984;311:562-5. 13. Fromowitz FB, Viola MV, Chao S, et aL ras p21 expression in the progression of breast cancer. Hum Pathol 1987;18:1268-75. 14. Clair T, Miller WR, Cho-Chung YS. Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer. Cancer Res 1987;47:5290-3. 15. Viola MV, Fromowitz FB, Oravez S, et al. Expression of ras oncogene p21 in prostate cancer. N Engl J Med 1986;314:133-7.
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MIYAHARA E T AL
16. Japanese Research Society for Cancer of Colon and Rectum. General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus. Part I. Clinical Classification. Jpn J Surg 1983;13: 557-73. 17. Furth ME, Davis LJ, Fleurdelys B, Scolnick EM. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and the cellular ras gene family. J Virol 1982;43: 294-304. 18. Shimizu K, Birnbaum D, Ruley MA, e t al. Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1. Nature 1983;304:497-500. 19. Tabin CJ, Bradley SM, Bargmann CI, e t al. Mechanism of activation of a human oncogene. Nature 1982;300:143-9. 20. Hsu S, Raine L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 1981;29:577-80. 21. Reddy EP, Reynolds RK, Santos E, Barbacid M. A point mutation is responsible for the acquisition of
22.
23.
24.
25.
26.
Dis Colon Rectum, December 1991
transforming properties by the 124 human bladder carcinoma oncogene. Nature 1982;300:149-52. Salmon DJ, DeKernion JB, Verma IM, Cline MJ. Expression of cellular oncogenes in human malignancies. Science 1984;224:256-62. Czerniak B, Herz F, Gorczyca W, Koss LG. Expression of ras oncogene p21 protein in early gastric carcinoma and adjacent gastric epithelia. Cancer 1989;64:1467-73. Hand PH, Thor A, Wunderlich D, Muraro R, Carso A, Schlom J. Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas. Proc Natl Acad Sci USA 1984;81:5227-31. Gallick GE, Kurzrock R, Kloetzer WS, Arlinghaus RB, Gutterman JU. Expression of p21 ras in fresh primary and metastatic colorectal tumors. Proc Natl Acad Sci USA 1985;82:1795-9. Czerniak B, Herz F, Koss LG, SchlomJ. ras oncogene p21 as a tumor marker in cytodiagnosis of gastric and colonic carcinomas. Cancer 1987;60:2432-6.
T
Address reprint requests to Dr. Miyahara:Department of Surgery I, Medical College of Oita, 1-1 Idaigaoka Hasamamachi, Oitagun, Oita 879-56,Japan. 1097
The object of the present study was to search for the overexpression of the ras o n c o g e n e in primary colorectal cancer.
MATERIALS A N D M E T H O D S Patients I n c l u d e d in this study were 45 Japanese patients with colorectal cancer (16 colon and 29 rectal) treated in our clinic. The m e a n age of the patients was 60.5 years (range, 33-84 years). Twenty-nine patients were men, and 16 were w o m e n . All patients u n d e r w e n t resection of the primary lesion with l y m p h a d e n e c t o m y (25 colectomy, 20 rectal amputation). Thirty-one of these patients underwent curative resection for primary colorectal cancers at the time of the initial surgery, whereas the remaining 14 (10 with liver metastasis, 3 with distant lymph n o d e metastasis, and 1 with peritoneal dissemination) u n d e r w e n t noncurative resection. H e p a t e c t o m y was p e r f o r m e d in five patients with a liver metastasis, and hepatic arterial cannulation was d o n e for the remaining five patients. E x t e n d e d l y m p h a d e n e c t o m y was p e r f o r m e d in three patients with distant lymph n o d e metastasis. Cisplatin was given intraperitoneally to o n e patient with peritoneal dissemination of the cancer. 5-Fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-FU (tegafur) or uracil/FT was prescribed orally for patients treated by curative resection. A combination c h e m o t h e r a p y of mitomycin C, Adriamycin (Farmitalia, Milan, Italy), and 5-FU was given to patients for w h o m noncurative resection was done. Clinicopathologic findings and evaluations of operative p r o c e d u r e s w e r e according to "General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus" established by the Japanese Research Society for Cancer of Colon TM
1098
MIYAHARA E T AL
and Rectum, ~6 and clinical staging was according to the Union Internationale Contre le Cancer TNM classification.
Dis Colon Rectum, December 1991
containing 0.01 percent H202 for 5 minutes, washed, and counterstained with hematoxylin.
Statistics Anti-ras p 2 1 M o n o c l o n a l A n t i b o d y Biotinylated anti-v-H-ras p21 antibody (Y13-259) was purchased from Oncogene Science, Inc. (Mineola, NY). This is a rat monoclonal antibody directed against the p21 ras gene translational product of the Harvey strain of rat sarcoma virus 17 and reacts with the p21 translational products of H-, K-, and N-c-ras human oncogenes. 18' 19
Immunohistochemical Staining of Tissues The avidin-biotin-complex immunoperoxidase assay method 2~ was used to stain the excised tissues. Four-micron sections were deparaffinized and immersed in methanol containing 0.3 percent H202 for 30 minutes to eliminate the endogenous peroxidase activity. The sections were then washed with phosphate-buffered saline (PBS), normal mouse serum was added, and the preparations were incubated for 20 minutes at room temperature. The slides were then incubated at 4~ for eight hours with biotinylated anti-v-H-ras p21 antibody (2 #g/ml), washed with PBS, and incubated at room temperature for 15 minutes after the addition of peroxidase-avidin complexes. After washing with PBS, the slides were treated with 0.05 percent diaminobenzidine in 0.05 M Tris-HC1 (pH 7.2)
Correlations between p21 overexpression and pathologic features were examined using the chisquared test or Fisher's exact test. Survival rate was estimated on the basis of the Kaplan-Meier method, and the generalized Wilcoxon test was used for analysis. Pvalues of less than 0.05 were considered to be statistically significant. RESULTS
Expression of p21 in Colorectal Cancer Tissues Two micrograms per milliliter of Y13-259 were used since the cytoplasm of the cancer cells was well stained, but the adjacent normal mucosa was less stained with this concentration (Fig. 1). The positivity of the staining was classified into two groups, based on the ratio of ras p21-positive cancer ceils; specimens containing over 50 percent of ras p21-positive cancer cells were classed as the positive group and those under 50 percent as the negative group. The intensity of the staining varied with each specimen. As shown in Table 1, 45 patients were divided into two groups: the positive group (14 patients; 31 percent) and the negative group (31 patients; 69 percent). Backgrounds of
Figure 1. Immunohistochemical staining of ras p21 with monoclonal antibody Y13-259 and the avidin-biotin-peroxidase complex technique. A. Positive case: peroxidase reaction is seen in the cytoplasm of cancer cells. B. Negative case. (Hematoxylin counterstain, x400.)
ras p21 EXPRESSION IN COLORECTAL CANCER
Vol. 34, No. 12
Table 1. ras p21 Overexpression in Colorectal Cancer ras p21 Overexpression No. of patients (%)
Positive
Negative
14 (31)
31 (69)
Age (years) Range Mean Sex Male (%) Female (%) Duration of symptoms __7 months (%)
Negative (n = 31)
P Value
42-84 33-79 63.6_+11.7 59.1_+11.8 0.113 9 (64) 5 (36) 3 (21) 3 (21) 4 (29) 4 (29)
ras p21 Overexpression
45 (100)
ras p21 Overexpression Positive (n = 14)
Table 3. ras p21 Overexpression and Tumor Size
Total
Table 2. ras p21 Overexpression and Clinical Characteristics
20 (65) 11 (35) 5 (16) 10 (32) 8 (26) 8 (26)
1099
Length (cm)* Widtht 0-30 (%) 31-60 (%) 61-100 (%)
Positive (n = 14)
Negative (n = 31)
5.5 + 1.4
4.6 _+ 1.9
0 (0) 2 (14) 9 (86)
5 (17) 7 (23) 19 (61)
P Value
0.0491"
0.189
* Mean longitudinal length of the tumor. t Significant difference. :[: Ratio of tumor-occupying width to entire circumference of intestinal wall. Table 4. ras p21 Overexpression and Histopathologic Findings
1.000
0.897
the patients were compared (Table 2). Differences between the two groups with regard to sex, mean age, and duration of symptoms before admission were not significant. Expression of p2 1 and Clinicopathologic Findings Correlations of the overexpression of ras p21 and clinicopathologic findings were also investigated. The longitudinal length of the tumor was significantly greater in the positive than in the negative group (Table 3). However, there was no significant difference between the two groups with regard to the ratio of tumor-occupying width to entire circumference of the intestinal wall. Histologic types were not related to the overexpression of ras p21 (Table 4). From the 14 patients of the ras p21-positive group, there was no specimen in which carcinoma was limitedto within the proper muscle layer (pro). In four specimens (29 percent) of this group, the malignant cells extended beyond the proper muscle layer but were not exposed to the serosal surface (ss) or the adventitial layer (al). Infiltration to the serosal or adventitial layer (s,a2) or to the neighboring tissue (s~,a,) was obvious in the other 10 specimens (71
ras p21 Overexpression
Histologic type* Well (%) Moderately (%) Poorly (%) Mucinous (%) Depth of invasionipm (%) ss,a~ (%) s,a2; s~,a,(%) Lymphatic invasion (+):l: (%) Venous invasion (+) (%)
P Value
Positive (n = 14)
Negative (n = 31)
10 (71) 3 (21) 1 (7) 0 (0)
23 (74) 6 (19) 0 (0) 2 (6)
0.369
0 (0) 24 (29) 10 (71) 14 (100)
4 (13) 16 (52) 11 (35) 18 (58)
0.060 0.005w
6 (43)
5 (16)
0.124
* Well: well-differentiated adenocarcinoma; Moderately: moderately differentiated adenocarcinoma; Poorly: poorly differentiated adenocarcinoma; Mucinous: mucinous carcinoma. 1 pm: cancer is confined to within proper muscular layer; ss,al: cancer invades serosa or adventitia to some extent; s,a2: cancer invades serosa or adventitia extensively; s~,ag cancer infiltrates neighboring tissue. ~:(+): Present. w Significant difference. percent). On the other hand, in the ras p21-negative group, there were only 11 of 31 specimens (35 percent) with invasion of s,a2 or si,ai (Table 4). Lymphatic vessel invasion of cancer cells was detected in all 14 specimens in the ras p21-positive group and in 18 of 31 (58 percent) in the negative group, with a statistically significant difference (P < 0.05). Venous invasion of cancer cells was present in 6 of 14 specimens (43 percent) in the ras
1100
MIYAHARA E T AL
p21-positive group and in 5 of 31 (16 percent) in the negative group. Venous invasion tended to be more frequent in specimens with ras p21 overexpression than in those without it, but with no statistical significance. Twenty-three patients had lymph node metastases and ten liver metasatases at the time of the initial surgery. There was a positive lymph node metastasis in 79 percent of those in the ras p21positive group but in only 39 percent of those in the negative group (Table 5). The incidence of liver metastasis in the ras p21-positive group was also higher than in the negative group. Clinical stages and operative curabilities in patients with or without overexpression of p21 are shown in Table 6. There was a significant difference with regard to TNM stage between the two groups. The rates of noncurative resection in the p21-positive group and in the p21-negative group were 64 percent and 16 percent, respectively. Table 5. ras p21 Overexpression and Metastasis ras p21 Overexpression
Lymph node metastasis Positive (%) Negative (%) Liver metastasis Positive (%) Negative (%)
P Value
Positive (n=14)
Negative (n=31)
11 (79) 3 (21)
12 (39) 19 (61)
0.029*
6 (43) 8 (57)
4 (13) 27 (87)
0.069
* Significant difference. Table 6. ras p21 Overexpression and Clinical Stage and Curability of Operation ras p21 Overexpression
Clinical stage* la, Ib, II (%) III, IV (%) Curability of operation:l: Curative (%) Noncurative (%)
P Value
Positive (n=14)
Negative (n=31)
2 (14) 12 (86)
17 (55) 14 (45)
0.022t
5 (36) 9 (64)
26 (84) 5 (16)
0.0041"
* TNM stage. 1" Significant difference. 1: Evaluation of operative procedure according to "General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus". 16
100
Dis Colon Rectum, December 1991
-•6-6--8-J----•
....... 6- rasop21 negative (n=31) .......
.._J
~
1 ........
i
1,
..... , ..... ,, 6 --L--u--t---t 59.7
~
S0
J
i
J
i
ras p21 positive (n = 14)
-
~-
0
I
1
I
I
i I
2 3 4 YEARSAFTEROPERATION
16.1 I
5
Figure 2. Survival curves of patients with resected colorectal cancer, with or without overexpression of ras p21. The difference was statistically significant (P < 0.05). Figure 2 shows overall survival curves of the groups, with or without the overexpression of ras p21. The five-year survival rates of the p21-positive and negative groups were 16.1 percent and 59.7 percent, respectively; the difference was statistically significant. DISCUSSION The ras oncogene family was seen to relate with tumor development, progression, and metastasis, either following activation of the gene resulting from a point mutation at amino acids 12 or 6119' 21 or following overexpression of the gene product, p21.12 15,22 Monoclonal antibodies to ras p21 can facilitate detection of the expressed ras p21 in human tissues. The antibody Y13-259 used in the present study and the RAP-512 are widely used to recognize the p21 product of human ras genes since there is a specific reaction with ras p21 in fixed tissues. 12' 17-19,23 Czerniak e t al. 23 noted that the intensity of staining and the proportion of positively reacting cells were similar with Y13-259 and RAP-5 in cases of gastric cancer. With regard to human colorectal cancer, there are conflicting reports on the relationship between ras p21 overexpression and tumor progression. 12'24-26 Immunohistochemical studies done using RAP antibodies showed that the increased ras p21 expression correlates with the depth of invasion of colonic carcinomas. 12' 24 On the other hand, Gallick e t al. 25 reported that relatively early Dukes' stages of tumors also have elevated levels of ras p21.
Vol. 34, No. 12
ras p21 EXPRESSION IN COLORECTAL CANCER
We observed that overexpression of ras p21 statistically related to clinicopathologic factors is considered to be closely linked to a p o o r prognosis, i . e . , size of tumor, lymphatic vessel invasion, and liver metastasis. T h e r e was a t e n d e n c y toward a relation, albeit not statistically, to the d e p t h of invasion, venous invasion, and liver metastasis. T h e r e was also a significant difference b e t w e e n the ras p21-positive group and negative group concerning long-term survival. Our present study clearly s h o w e d that the prognosis of patients with ras p21 overexpression was p o o r e r than that of those without it. The lower survival rate in the p21-positive patients was based on the finding that patients in the positive group had a higher rate of noncurative resection at the initial surgery. Factors evaluated as noncurative were liver metastasis, distant lymph n o d e metastasis, or peritoneal dissemination. Thus, colorectal cancer highly expressing ras p21 has a p o t e n t biologic malignancy potential, as reflected by tumor progression, metastasis, and a p o o r prognosis. Various clinicopathologic prognostic factors include histologic type, d e p t h of invasion, and vessel involvement. 1-4 We f o u n d that overexpression of ras p21 c o r r e s p o n d e d with the pathologic progression and clinical stage of the cancer. Therefore, i m m u n o h i s t o c h e m i c a l d e t e c t i o n of overexpression with Y13-259, especially in preoperatively b i o p s i e d tissues, should b e c o n s i d e r e d for evaluation of the a d v a n c e m e n t of the cancer. O n c e this e v i d e n c e has b e e n obtained, suitable treatment can be prescribed. CONCLUSION Detection of ras p21 overexpression in colorectal cancer by the avidin-biotin-complex i m m u n o p e r oxidase assay m e t h o d can serve as a useful tool for clinical evaluation of the potential malignancy of tumors in patients. ACKNOWLEDGMENT The authors thank M. Ohara for critical comments.
REFERENCES 1. olson RM, Perencevich NP, Malcolm AW, Chaffey JT, Wilson RE. Pattern of recurrence following cu-
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rative resection adenocarcinoma of the colon and rectum. Cancer 1980;45:2969-74. 2. Eisenberg B, Decosse JI, Hartford F, Michalek J. Carcinoma of the colon and rectum: the natural history reviewed in 1704 patients. Cancer 1982; 49:1131-4. 3. Muraoka Y, Maetani S, Tobe T. Statistical analysis of pathological factors influencing prognosis of colorectal carcinoma. J Jpn Surg Soc 1988;89:181-91. 4. Kotanagi H, Nagasawa O, Niwa M, Takahashi M, Narisawa T, Koyama K. Prediction of the hematogenic metastasis and local recurrence of rectal cancer by quantification of the clinico-pathological factors. J Jpn surg Soc 1988;89:1022-7. 5. Papageorge A, Lowy DR, Scolnick EM. Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol 1982;44:509-19. 6. Hurley JB, Simon MI, Teplow DB. Homologies between signal transducing G proteins and gene products. Science 1984;226:860-2. 7. Thorgeirsson UP, Turpeenniemi-Hujanen T, WilliamsJE, et al. NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol 1985;5:259-62. 8. Bernstein SC, Winberg RA. Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA. Proc Natl Acad Sci USA 1985;82:1726-30. 9. Spandidos DA, Wilkie NM. Malignant transformation of early passage rodent cells by a single mutated human oncogene. Nature 1984;310:469-75. 10. Muschel RJ, WilliamsJE, Lowy DR, Liotta LA. Harvey ras induction of metastatic potential depends upon oncogene activation and the type of recipient cell. Am J Pathol 1985; 121:1-8. 11. Bradley MO, Kraynak AR, Storer RD, Gibbs JB. Experimental metastasis in nude mice of NIH3T3 cells containing various ras genes. Proc Natl Acad Sci USA 1986;83:5277-81. 12. Thor A, Hand PH, Wunderlich D, Caruso A, Muraro R, Schlom J. Monoclonal antibodies define differential ras gene expression in malignant and benign colonic disease. Nature 1984;311:562-5. 13. Fromowitz FB, Viola MV, Chao S, et aL ras p21 expression in the progression of breast cancer. Hum Pathol 1987;18:1268-75. 14. Clair T, Miller WR, Cho-Chung YS. Prognostic significance of the expression of a ras protein with a molecular weight of 21,000 by human breast cancer. Cancer Res 1987;47:5290-3. 15. Viola MV, Fromowitz FB, Oravez S, et al. Expression of ras oncogene p21 in prostate cancer. N Engl J Med 1986;314:133-7.
1102
MIYAHARA E T AL
16. Japanese Research Society for Cancer of Colon and Rectum. General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus. Part I. Clinical Classification. Jpn J Surg 1983;13: 557-73. 17. Furth ME, Davis LJ, Fleurdelys B, Scolnick EM. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and the cellular ras gene family. J Virol 1982;43: 294-304. 18. Shimizu K, Birnbaum D, Ruley MA, e t al. Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1. Nature 1983;304:497-500. 19. Tabin CJ, Bradley SM, Bargmann CI, e t al. Mechanism of activation of a human oncogene. Nature 1982;300:143-9. 20. Hsu S, Raine L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 1981;29:577-80. 21. Reddy EP, Reynolds RK, Santos E, Barbacid M. A point mutation is responsible for the acquisition of
22.
23.
24.
25.
26.
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transforming properties by the 124 human bladder carcinoma oncogene. Nature 1982;300:149-52. Salmon DJ, DeKernion JB, Verma IM, Cline MJ. Expression of cellular oncogenes in human malignancies. Science 1984;224:256-62. Czerniak B, Herz F, Gorczyca W, Koss LG. Expression of ras oncogene p21 protein in early gastric carcinoma and adjacent gastric epithelia. Cancer 1989;64:1467-73. Hand PH, Thor A, Wunderlich D, Muraro R, Carso A, Schlom J. Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas. Proc Natl Acad Sci USA 1984;81:5227-31. Gallick GE, Kurzrock R, Kloetzer WS, Arlinghaus RB, Gutterman JU. Expression of p21 ras in fresh primary and metastatic colorectal tumors. Proc Natl Acad Sci USA 1985;82:1795-9. Czerniak B, Herz F, Koss LG, SchlomJ. ras oncogene p21 as a tumor marker in cytodiagnosis of gastric and colonic carcinomas. Cancer 1987;60:2432-6.
