Abstract
Purpose
The significance of p34cdc2 expression in human tumors has not been fully explained. The aim of the current study was to elucidate the clinicopathologic significance of immunohistochemical p34cdc2 expression in carcinoma of the colon and rectum.
Methods
The immunohistochemical expression of p34cdc2 was examined in 90 consecutive colorectal tumor cases, and p34cdc2 expression and the clinicopathologic features of the patients and their tumors were compared.
Results
Lymph node metastasis was significantly more frequent in tumors expressing p34cdc2 (47.8%, 11 of 23 tumors) than in tumors not expressing p34cdc2 (22.4%, 15 of 67 tumors; P=0.020). Multivariate analysis demonstrated that tumor depth (P=0.008) and p34cdc2 expression (P=0.022) were independently associated with lymph node metastases of colorectal carcinomas.
Conclusions
The immunohistochemical expression of p34cdc2 is independently associated with lymph node metastasis in colorectal carcinoma.
Keywords: Colorectal carcinoma, p34cdc2, Immunohistochemistry, Lymph node metastasis
Introduction
Cell cycle-mediating cyclins play a role in controlling many of the steps in cell cycle progression, especially in the G1/S and G2/M transitions, by forming complexes with such kinases as cdk (cyclin-dependent kinase) and cdc (cell division cycle) kinase (Morla et al. 1989; Pines and Hunter 1989; Jessus and Beach 1992). The human Cdc2+ gene codes a serine/threonine kinase. Cdc2 protein, that is p34cdc2, exerts a protein kinase activity by constructing complexes with cyclin A, cyclin B, and p13suc1 (Brizuela et al. 1987; Draetta et al. 1987; Nigg 1995) and is therefore known as an active subunit of the M-phase promoting factor (MPF) and the M-phase specific histone H1 kinase (Arion et al. 1988). Moreover, p34cdc2 also plays a role in phosphorylating the retinoblastoma gene product, which regulates the cell cycle by restricting DNA replication (Goodrich et al. 1991) at G2/M transition (Lees et al. 1991).
The levels of cyclin B protein change during cell cycle and increase as the cycle progresses toward the M phase; almost all of the protein is degraded after the end of the M phase. On the other hand, p34cdc2 levels remain fairly constant during the cell cycle; the protein is known to exert a function in M phase regulation only by forming a complex with cyclin B (Lew and Kornbluth 1996).
Although the definitive significance of cyclin expression at the G2/M transition as an indicator of malignant potential of human tumors has been clearly demonstrated in many investigations (Furihata et al. 1996; Volm et al. 1997; Murakami et al. 1999; Allan et al. 2000; Florenes et al. 2001; Nozoe et al. 2002), the implications of the immunohistochemical expression of p34cdc2 have not been fully explored.
The aim of this study was to elucidate the clinicopathologic significance of the immunohistochemical expression of p34cdc2 in carcinoma of the colon and rectum.
Materials and methods
Patients and sample collection
Specimens were collected from 90 patients with colorectal carcinoma who had undergone surgery at our department or its affiliated hospitals between January 1984 and May 2000. The specimens were embedded in paraffin, and representative sections of each specimen were stained with hematoxylin and eosin.
Patient were followed up until death, and only those who died of colorectal carcinoma were regarded as having died of tumor-related causes. The follow-up interval after surgery ranged from 79 days to 16 years and 11 months, with a mean of 6 years and 2 months.
The clinicopathologic results were evaluated according to the general rules for clinical and pathologic studies on cancer of the colon, rectum, and anus outlined by the Japanese Research Society for Cancer of the Colon and Rectum (Japanese Research Society for Cancer of the Colon and Rectum 1983). The depths of the tumors were described in accordance with the TNM classification of malignant tumors prescribed by the International Union Against Cancer (Sobin and Wittekind 1997). The pathologic stages of the tumors were defined using Dukes' classification criteria (Dukes and Bussey 1958).
Immunohistochemical expression of p34cdc2
Four-micrometer-thick sections sliced from paraffin-embedded specimen were prepared on the slide glasses precoated with silane. After removing the paraffin with xylene and washing in a graded series of ethanol, the sections were placed in Tris-buffered saline (TBS) for 10 min. Endogenous peroxidase activity was blocked for 10 min in blocking solution, then the slides were placed in TBS. The sections were incubated with TBS including 1% concentration of bovine serum albumin for 20 min to block nonspecific binding of the immunoreagents. After washing in TBS, the sections were incubated with 1:100 diluted mouse anti-human monoclonal p34cdc2 antibody (sc-54, Santa Cruz Biotechnologies, Santa Cruz, CA). All incubations proceeded overnight at 4°C. After washing in TBS, an immunoperoxidase staining was performed by an EnVision antibody complex method (Sabattini et al. 1998; Kammerer et al. 2001) using ENVISION kit (DAKO. Ltd., Tokyo, Japan). Finally, the localization of FasL was visualized with diaminobenzidine tetrahydrochloride.
Specimens showing p34cdc2 expression in the nuclei of more than 50% of examined cancer cells were considered to have a positive response to p34cdc2.
Statistical analysis
Data were expressed as the mean ± standard error of the mean. Statistical comparisons were made with the chi-square test, Student's t test, and the Mann-Whitney U test. Cumulative survival rates were calculated with the Kaplan-Meier method and the survival curves were examined by the Mantel-Cox method. Clinicopathologic factors independently associated with lymph node metastasis were tested by means of stepwise logistic regression analysis. The multivariate survival analysis was calculated according to Cox's proportional hazards model in the forward stepwise manner. A P value of less than 0.05 was considered statistically significant.
Results
The immunohistochemical expression of p34cdc2 in colorectal carcinoma is demonstrated in Fig. 1. Comparison of p34cdc2 expression and the clinicopathologic characteristics is shown in Table 1. Patients with colorectal carcinomas expressing p34cdc2 (60.2±14.8 years) were significantly younger than those with tumors exhibiting no p34cdc2 expression (66.4±10.8 years; P=0.034). The incidence of lymph node metastasis was significantly higher in tumors expressing p34cdc2 (47.8%, 11 of 23 tumors) than in tumors with no p34cdc2 expression (22.4%, 15 of 67; P=0.020). No significant difference was observed with respect to tumor-related factors other than lymph node metastasis. Multivariate analysis demonstrated that depth of invasion (P=0.008) and p34cdc2 expression (P=0.022) were independently associated with lymph node metastasis in colorectal carcinomas (Table 2).
Fig. 1A, B.
Immunohistochemical expression of p34cdc2 in colon carcinoma. Its expression in the nuclei of the carcinoma cells is demonstrated. A Photo at lower magnification, ×100; B photo at higher magnification, ×200
Table 1.
p34cdc2 expression and clinicopathologic characteristics
| p34cdc2 positive | p34cdc2 negative | P | |
|---|---|---|---|
| (n=23) | (n=67) | ||
| Gender | |||
| Male | 11 (47.8) | 37 (55.2) | 0.540 |
| Female | 12 (52.2) | 30 (44.8) | |
| Age (yrs) (SEM) | 60.2±14.8 | 66.4±10.8 | 0.034 |
| Location of tumors | |||
| Cecum and ascending colon | 1 (4.3) | 12 (17.9) | 0.472 |
| Transverse colon | 0 | 5 (7.5) | |
| Descending colon | 2 (8.7) | 2 (3.0) | |
| Sigmoid colon | 10 (43.5) | 26 (38.8) | |
| Rectum | 10 (43.5) | 22 (32.8) | |
| Size of tumor (cm) (SEM) | 5.6±2.7 | 4.87±2.1 | 0.139 |
| Pathological type | |||
| Well diff. adenoca. | 12 (52.2) | 37 (55.2) | 0.251 |
| Moderately diff. adenoca. | 9 (39.1) | 29 (43.3) | |
| Poorly diff. adenoca. | 2 (8.7) | 1 (1.5) | |
| Depth of invasion | |||
| T1 | 1 (4.3) | 8 (11.9) | 0.379 |
| T2 | 11 (47.8) | 29 (43.3) | |
| T3 | 8 (34.8) | 27 (40.3) | |
| T4 | 3 (13.1) | 3 (4.5) | |
| Lymph nodes metastasis | |||
| Positive | 11 (47.8) | 15 (22.4) | 0.020 |
| Negative | 12 (52.2) | 52 (77.6) | |
| Lymphatic permeation | |||
| Positive | 6 (26.1) | 16 (23.9) | 0.833 |
| Negative | 17 (73.9) | 51 (76.1) | |
| Venous invasion | |||
| Positive | 9 (39.1) | 15 (22,4) | 0.117 |
| Negative | 14(60.9) | 52 (77.6) | |
| Distant metastasis | |||
| Positive | 2 (8.7) | 6 (9.0) | 0.970 |
| Negative | 21 (91.3) | 61 (91.0) | |
| Dukes' classification | |||
| A | 1 (4.4) | 7 (10.4) | 0.278 |
| B | 11 (47.8) | 41 (61.2) | |
| C | 9 (39.1) | 13 (19.4) | |
| D | 2 (8.7) | 6 (9.0) |
Values in the parentheses are percentages
SEM, Standard error of the mean; diff., differentiated; adenoca., adenocarcinoma
Table 2.
Factors independently associated with lymph node metastasis
| Standard error | Odds ratio (95% confidence interval) | P | |
|---|---|---|---|
| Depth of invasion | 0.642 | 5.43 (1.54–19.1) | 0.008 |
| p34cdc2 expression | 0.614 | 4.10 (1.23–13.7) | 0.022 |
Survival rates at 1, 3 and 5 years for patients with colorectal carcinomas expressing p34cdc2 were 91.3%, 82.2%, and 77.6%, respectively; the corresponding survival rates for patients whose tumors did not express p34cdc2 were 90.9%, 80.3%, and 78.7%, respectively. Survival rates for the two groups of patients were not significantly different (P=0.761; Fig. 2). Multivariate analysis revealed that lymph node metastasis (P=0.0004) and tumor stage determined on the basis of Dukes' classification (P<0.0001) were independent prognosticators in colorectal carcinoma (Table 3).
Fig. 2.
Survival curves of patients with colorectal carcinoma with or without p34cdc2 expression. No significant difference was observed between the prognoses of patients with colorectal tumors with p34cdc2 expression (solid line) and patients who had tumours without p34cdc2 expression (dotted line) (P=0.761)
Table 3.
Factors independently associated with prognosis
| Variable | Regression coefficient | Standard error | Odds ratio (95% confidence interval) | P |
|---|---|---|---|---|
| Lymph node metastasis | 2.997 | 0.847 | 20.03 (3.809–105.3) | 0.0004 |
| Lymphatic permeation | 0.500 | 0.462 | 1.647 (0.667–4.082) | 0.279 |
| Venous invasion | 0.004 | 0.506 | 1.004 (0.372–2.708) | 0.994 |
| Tumor stage | 3.735 | 0.855 | 41.67 (7.813–250.0) | <0.0001 |
| p34cdc2 expression | 0.039 | 0.482 | 1.040 (0.397–2.725) | 0.937 |
Discussion
Of the cyclin-kinase complexes, incorporation of cyclin B and p34cdc2 kinase is the best characterized form and the complex is known to play a pivotal role in the progression of the G2/M transition. Because expression of such G2/M cyclins as cyclin A (Furihata et al. 1996; Volm et al. 1997; Florenes et al. 2001) and cyclin B1 (Murakami et al. 1999; Allan et al. 2000; Nozoe et al. 2002) is correlated with the biological behavior of the human tumor, reflecting a poorer prognosis for patients, it seems to be quite natural to focus on the expression of p34cdc2, which functions by forming complexes with G2/M cyclins. However, the number of studies that have investigated the clinicopathologic significance of p34cdc2 expression in human tumors is unexpectedly small (Yasui et al. 1993; Ito et al. 2000; Winters et al. 2001).
By viewing the function of the p34cdc2/cyclin B1 complex in the G2/M transition, one can readily arrive at the prediction that p34cdc2 expression may also be related to the malignant potential of human tumors, including colorectal carcinoma (Yasui et al. 1993; Kallakury et al. 1999; Ito et al. 2000). Yasui et al. (1993) described correlation of p34cdc2 expression in colorectal carcinoma with the expression of proliferating cell nuclear antigen, as well as a relationship between high cdc2 kinase activity and p53 expression. Their results may demonstrate a possible correlation of p34cdc2 expression with the development and proliferation of colorectal carcinoma.
The most outstanding finding shown in the current study is a significant correlation of immunohistochemical expression of p34cdc2 with lymph node metastasis in colorectal carcinoma. Therefore p34cdc2 expression may at least be an indicator of the malignant potential of colorectal carcinoma. However, lymph node metastasis and tumor stage determined based on Dukes' classification but not p34cdc2 expression were found to be an independent prognosticator in colorectal carcinoma. Therefore the expression of p34cdc2 did not reflect the worse prognosis of the patients with colorectal carcinoma.
In our institution, the biological significance of cyclin B1 expression in human gastrointestinal carcinomas has been investigated. We have demonstrated that nuclear dominant expression of cyclin B1 could be an independent prognosticator in esophageal squamous cell carcinoma (Nozoe et al. 2002) and that a significant correlation exists between cytoplasmic expression of cyclin B1 and lower incidence of lymph node metastasis in gastric and colorectal carcinomas (Korenaga et al. 2002; Yasuda et al. 2002). These results suggest that cyclin B1 protein located in the nuclei exerts a function in cell cycle progression. Hence, the expression of p34cdc2 in the nuclei of colorectal carcinoma cells, which was defined as positive p34cdc2 expression in colorectal carcinoma cases enrolled in the current study, is considered to function in association with cyclin B1 at the G2/M checkpoint.
In conclusion, immunohistochemical expression of p34cdc2 was found to be independently associated with lymph node metastasis in colorectal carcinoma. We believe that our results indicate the need for further studies into the functions of cell cycle-related proteins in modulating the G2/M checkpoint, which controls the mitotic potential of colorectal carcinoma cells.
Acknowledgements
This study was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (Frontier Research Grant).
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