Prognostic Value of Cyclin B1 Protein Expression in Colorectal Cancer

Anatomic Pathology / CYCLIN B1 IN COLORECTAL CANCER

Prognostic Value of Cyclin B1 Protein Expression in Colorectal Cancer Heike Grabsch, MD,1* Kristina Lickvers, MD,2* Olaf Hansen, MD,3 Shinsuke Takeno, MD,4 Reinhart Willers, PhD,5 Wolfgang Stock, MD,3 Helmut E. Gabbert, MD,1 and Wolfram Mueller, MD6 Key Words: Cyclin B1; Colorectal cancer; Prognosis; Cell cycle regulation DOI: 10.1309/54H4Q88A1UBBWPTE

Abstract We used immunohistochemical analysis to study the expression and prognostic impact of cyclin B1, a key molecule for G2-M phase transition during the cell cycle, in a series of 342 curatively resected colorectal carcinomas. In 269 tumors (78.7%), high expression of cyclin B1 in more than 10% of tumor cells was observed, but there was no association between cyclin B1 expression and histopathologic tumor features. Univariate analysis revealed no impact on disease-free and overall survival. Multivariate analysis revealed pT and pN categories, extramural blood vessel invasion, and low-grade tumor cell differentiation as independent prognostic predictors for overall survival, and pT and pN categories and tumor site for disease-free survival. According to our results, high expression of cyclin B1 is a frequent and early event in colorectal carcinomas. However, cyclin B1 expression is neither a predictor of prognosis in patients with colorectal cancer nor a suitable tool for identifying subgroups of patients at higher risk for disease recurrence.

Uncontrolled proliferation is one of the hallmarks of malignant cells.1 In normal cells, progression through the cell cycle is regulated by specific cyclins and cyclin-dependent kinases.2 Specifically, the onset of mitosis is regulated by the activation of a complex of cyclin B1 and cdc2, originally defined as the M phase promoting factor.3,4 Because cdc2 generally is present abundantly throughout the cell cycle, synthesis and deactivation of its positive regulatory subunit, cyclin B1, seems to be the main mechanism for controlling the activity of the cyclin B1–cdc2 complex. In human cells, cyclin B1 expression starts at the end of the S phase with a peak at the G2M transition,5 and activation of the cyclin B1–cdc2 complex leads to chromosome condensation, destruction of the nuclear envelope, and assembly of the mitotic spindle.6,7 Its activity is regulated by different phosphorylation events resulting in inhibition of kinase activity by members of the Wee1/Mik1 family of protein kinases8,9 or activation mediated by CDC25B and CDC25C counteracting Wee1 activity.10,11 Another control mechanism for cyclin B1–cdc2 activity is the localization to different subcellular compartments. Initially located in the cytoplasm during the S and G2 phases, cyclin B1 is translocated to the nucleus at the beginning of mitosis, mediating its biologic activity.12,13 Thus, controlling the G2-M phase checkpoint by cyclin B1–cdc2 complexes seems to have a key role in the regulation of the correct timing of mitotic entry that is essential for maintenance of genomic integrity. As a consequence, inappropriate expression, subcellular localization, or both of cyclin B1 might result in premature entry into mitosis, uncontrolled cell proliferation, and neoplastic transformation. Expression of cyclin B1 has been reported in breast, prostate, and oral cancers.14-16 Furthermore, expression of cyclin B1 was shown to Am J Clin Pathol 2004;122:511-516

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DOI: 10.1309/54H4Q88A1UBBWPTE

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Grabsch et al / CYCLIN B1 IN COLORECTAL CANCER

be associated with poorer survival of patients with squamous cell carcinomas of the esophagus,17,18 tongue,19 and lung.20 Concerning the expression of cyclin B1 in colorectal carcinomas, so far, to our knowledge, there is only 1 report on 41 tumors revealing higher expression of cyclin B1 in the tumor tissue than in the corresponding normal colon tissue for the majority of investigated cases.21 However, to our knowledge, there are no data available on the putative prognostic role of cyclin B1 expression in colorectal carcinomas. Therefore, our aims were to study the expression and subcellular localization of cyclin B1 in a series of 342 colorectal carcinomas by immunohistochemical analysis and to correlate the results with histopathologic variables and with disease-free and overall survival.

Materials and Methods Patients The present study is based on a series of 342 colorectal carcinomas from 330 patients who underwent potentially curative (R0) resection22 in the Department of Surgery, Marien Hospital, Düsseldorf, Germany, between January 1990 and December 1995. Survival data were available for all patients, including information on recurrence of disease. The median follow-up time was 4.2 years (range, 5 months to 11.4 years). Postoperatively, 14 patients received chemotherapy, 12 received radiotherapy, and 7 patients received both. Of the 342 tumors, 75 (21.9%) were located in the cecum or ascending colon; 35 (10.2%) at the hepatic flexure, transverse colon, or splenic flexure; 109 (31.9%) in the descending or sigmoid colon; and 123 (36.0%) in the rectosigmoid junction or rectum. Of 330 patients, 149 (45.2%) were men and 181 (54.8%) were women. The mean age was 68.8 years (range, 28-88 years). At the end of the follow-up period, local disease recurrence was noted in 25 patients, distant metastasis in 49, and both in 8. Pathologic Review Histologic sections from all 342 tumors were reviewed without knowledge of the clinical outcome. An average of 4 sections per tumor (range, 3-6 sections) was prepared, and paraffin sections were stained routinely with H&E and with elastic van Gieson for detection of blood vessel involvement. To compare the prognostic significance of cyclin B1 expression with other known prognostic parameters, the following morphologic details were recorded: depth of invasion (pT category), lymph node involvement (pN category), grade of tumor differentiation according to the World Health Organization,23 lymphatic vessel invasion, and blood vessel invasion (categorized into intramural, ie, submucosa or muscularis propria, and extramural, ie, subserosa, invasion). Tissue 512 512

Am J Clin Pathol 2004;122:511-516 DOI: 10.1309/54H4Q88A1UBBWPTE

for histopathologic diagnosis of distant metastasis (pM category) could be obtained for only 6 patients. Therefore, this variable was excluded from further analysis. Immunohistochemical Analysis From each patient, at least 1 representative tumor block including tumor-associated nonneoplastic mucosa was examined by immunohistochemical analysis. Immunohistochemical detection of cyclin B1 was performed with a monoclonal antibody (clone 7A9; Novocastra Laboratories, Newcastle upon Tyne, England) after heat antigen retrieval in a pressure cooker for 5 minutes in EDTA buffer (pH 8.0). The primary antibody was diluted (1:100) with antibody diluent (Zymed Laboratories, San Francisco, CA), and the tissue sections were incubated overnight at 4°C. Detection was performed using a nonbiotin amplification system according to the instructions of the manufacturer (NBA Kit, Zymed Laboratories) and AEC-Ready-to-Use (BioGenex, San Ramon, CA). Mayer haemalaun was used for counterstaining. Normal tonsils or lymph nodes were used as positive control tissue samples. Tumor-associated nonneoplastic colon mucosa served as an additional internal positive control sample. Negative control experiments were performed by omitting the primary antibody. Evaluation of Cyclin B1 Expression All slides were evaluated without knowledge of the clinical outcome. Tumor cells displaying cytoplasmic or nuclear staining were considered positive. Expression of cyclin B1 was categorized semiquantitatively into the following 4 groups: 1, fewer than 10% positive tumor cells; 2, 10% to 25% positive tumor cells; 3, 26% to 50% positive tumor cells; 4, more than 50% positive tumor cells. The staining intensity of the tumor cells was not considered. To determine interobserver variability, 100 randomly chosen tumors were evaluated independently by 2 pathologists (K.L., W.M.). There was full agreement in 87.0% of these cases. In the remaining cases, the 2 observers’ judgments differed by only 1 category. If the 10% level was used to discriminate between tumors with low and high expression of cyclin B1, discordance was found in only 3 cases. Statistical Analysis For statistical analysis, the SAS software package (SAS Institute, Cary, NC) was used, and comparisons of the distributions of cyclin B1 expression for different groups were performed using the χ2 test. Analyses of survival were performed using the Kaplan-Meier method,24 and differences between the patient groups were tested by using the log-rank test.25 Prognostic relevance was verified by applying univariate and multivariate Cox regression analysis. P values less than .05 were considered significant. © American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE

Results Cyclin B1 Expression in Normal Colorectal Mucosa In normal colorectal mucosa, expression of cyclin B1 was observed in all cases studied. The staining was located in the cytoplasm, and positive cells were found only at the proliferative zone in the lower third of the crypts. In all cases, the number of positive normal colonic epithelial cells did not exceed 10% of all normal cells at the proliferative zone of the crypt base (a representative case is shown in the inset) ❚Image 1A❚.

Cyclin B1 Expression in Colorectal Cancer In tumor tissues, immunoreactivity for cyclin B1 was observed in all 342 colorectal carcinomas cases and showed a cytoplasmic staining pattern. Strong intratumoral and intertumoral heterogeneity was noted, and the number of cyclin B1–positive tumor cells ranged from 1% to 80% ❚Image 1B❚ (Image 1A). Only a few tumor cells distributed randomly throughout the tissue samples showed combined cytoplasmic and nuclear cyclin B1 expression; no tumor cells showed nuclear staining only. The numbers and percentages of tumors staining positively for cyclin B1 are given in ❚Table 1❚.

A

B

C

D

❚Image 1❚ Cyclin B1 expression in colorectal carcinomas. A and B, Areas of an advanced colon carcinoma showing strong cytoplasmic staining with marked intratumoral heterogeneity (A, inset, representative case of cyclin B1 expression in tumorassociated normal colon mucosa located at the proliferative pool at the lower third of normal colon crypts, ×200) (A, ×400; B, ×400). C, Mitotic tumor cells showing cyclin B1 expression adjacent to a cyclin B1–negative mitosis (×630). D, Strong cyclin B1 expression (>50% of tumor cells) in carcinoma cells (right) adjacent to preexisting adenomatous glands showing cyclin B1 expression in only a few tumor cells (left) (×200).

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Looking at different areas of the tumor specimens revealed no differences in the number and distribution of cyclin B1–positive tumor cells between superficial and central parts of the tumor tissue or tumor areas at the tumor invasion front. Cyclin B1 expression was detected in the majority of mitotic cells; however, a few cyclin B1–negative mitotic tumor cells also were observed in every case ❚Image 1C❚. In 12 cases, tumor slides included parts of preexisting adenomas (all low-grade dysplasias) adjacent to the invasive carcinoma tissue. In all these cases, only a few adenoma cells showed cyclin B1 expression; in most of these cases, the adjacent invasive carcinoma showed high cyclin B1 expression ❚Image 1D❚. Correlation With Clinicopathologic Variables Because there were no differences among the 3 groups of tumors with more than 10% of cyclin B1–positive tumor cells concerning their association with clinicopathologic variables, for further statistical analysis, groups 2, 3, and 4 were summarized as tumors with high cyclin B1 expression. Accordingly, high cyclin B1 expression was found in 269 tumors (78.7%), and low cyclin B1 expression was observed in 73 tumors (21.3%). Analyzing the correlation between cyclin B1 expression and histopathologic data (depth of invasion [pT category], lymph node involvement [pN category], blood vessel and lymphatic vessel invasion, and grade of tumor cell differentiation) revealed no significant differences ❚Table 2❚. In addition, the frequency of high cyclin B1 expression did not differ significantly between age groups or by sex, and it did not correlate with tumor site (data not shown). Survival Analysis Univariate analysis based on the log-rank test revealed no statistically significant differences between patients with tumors with high cyclin B1 expression and those with low cyclin B1 expression in disease-free and overall survival rates ❚Figure 1❚. This also was true when tumors were analyzed separately according to the 4 scoring groups and when other cutoff points of cyclin B1 expression (20%, 30%, and up to 50%) were calculated. Furthermore, no significant influence on prognosis was detected when subgroups of patients with

❚Table 1❚ Distribution of Cyclin B1 Expression in 342 Colorectal Carcinomas Percentage of Positive Tumor Cells 50

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No. (%) of Tumors 73 (21.3) 99 (28.9) 120 (35.1) 50 (14.6)


different pT categories, pN categories, or tumor stage were analyzed separately. Multivariate analysis based on Cox regression analysis confirmed a statistically significant relationship between shorter overall survival and high pT category (P < .001), lymph node involvement (pN category, P < .001), extramural blood vessel invasion (P < .030), and low-grade tumor cell differentiation (P < .0181). For disease-free survival, pT category (P < .002), pN category (P< .001), and tumor localization (rectum and rectosigmoid, P < .005) were verified as independent prognostic variables (data not shown). However, no statistically significant relationship was observed between overall survival or disease-free survival and cyclin B1 expression.

Discussion The regulation of orderly cell cycle progression is essential for the ability of cells to maintain genomic integrity that is vital for cell survival and controlled proliferation. The cyclin B1–cdc2 complex seems to have a key role in regulating G2-M phase transition and the correct onset of mitosis, while its deregulation might lead to uncontrolled cell proliferation and loss of genomic stability. In fact, expression of

❚Table 2❚ Cyclin B1 Expression and Correlation With Different Histomorphologic Variables in 342 Colorectal Carcinomas* Cyclin B1 Expression Variable pT category pT1 (n = 25) pT2 (n = 67) pT3 (n = 223) pT4 (n = 27) pN category pN0 (n = 224) pN1 (n = 75) pN2 (n = 43) Stage I (n = 77) II (n = 145) III/IV† (n = 120) Grade 1‡/2 (n = 262) 3/4§ (n = 80) Blood vessel invasion|| Negative (n = 314) Intramural (n = 13) Extramural (n = 15) Lymphatic vessel invasion Negative (n = 249) Positive (n = 93)

Low

High

4 (16) 12 (18) 49 (22.0) 8 (30)

21 (84) 55 (82) 174 (78.0) 19 (70)

46 (20.5) 17 (23) 10 (23)

178 (79.5) 58 (77) 33 (77)

13 (17) 32 (22.1) 28 (23.3)

64 (83) 113 (77.9) 92 (76.7)

57 (21.8) 16 (20)

205 (78.2) 64 (80)

67 (21.3) 2 (15) 4 (27)

247 (78.7) 11 (85) 11 (73)

57 (22.9) 16 (17)

192 (77.1) 77 (83)

P .5616

.8786

.5377

.7373 .7679

.2534

*

Data are given as number (percentage). Stage IV, 6 tumors. ‡ Grade 1, 4 tumors. § Grade 4, 2 tumors. || Intramural, submucosa or muscularis propria; extramural, subserosa. †

© American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE

A

B

100

Disease-Free Survival (%)

Overall Survival (%)

100

50

0

50

100

150

75

50

25

0

50

100

150

Time (mo)

Time (mo)

❚Figure 1❚ Overall (A) and disease-free (B) survival rates for 330 patients with colorectal cancer with tumors with more than 10% of cyclin B1–positive tumor cells (n = 261; dotted line) and those with fewer than 10% of cyclin B1–positive tumor cells (n = 69; solid line). A, P = .1288; log-rank test. B, P = .5611; log-rank test.

cyclin B1 has been reported in a variety of human malignant neoplasms, including carcinomas of the breast, lung, esophagus, oral cavity, and prostate14-20 and in mucosa-associated lymphoid tissue lymphomas.26 Furthermore, at least in squamous cell carcinomas of the lung, oral cavity, and esophagus, a poor prognostic impact for cyclin B1 expression was observed.17-20 In colorectal cancer, however, cyclin B1 expression has not been studied in depth. The present study revealed that 269 (78.7%) of 342 colorectal carcinomas exhibited high expression of cyclin B1. It might be speculated that this increased expression of cyclin B1 only reflects increased cell proliferation. However, because only a subset of proliferating cells at the bases of the colonic crypts exhibited positive staining for cyclin B1 and only a small number of cells were positive for cyclin B1 within the adenomatous tissue, which is known to have high proliferative activity, it might be suggested that high expression of cyclin B1 is not just a simple consequence of enhanced cell proliferation, but rather an indicator of disturbed cell cycle progression at the G2-M checkpoint in colorectal carcinomas. This view is supported by investigations on breast27 and lung20 cancer that showed only weak association between proliferative activity (ie, Ki-67 expression) of tumor cells and cyclin B1 expression. In the present study, cyclin B1 expression was observed in the cytoplasm in the vast majority of colon cancer cells; only a small number of tumor cells showed additional cyclin B1 expression in the nucleus. Cyclin B1 localization in the cytoplasm has been described previously in lung,20 breast,14 and prostate15 cancers. In normal human cells,

cyclin B1–cdc2 complexes accumulate in the cytoplasm during late S-G2 phase and have to be translocated into the nucleus to initiate mitosis.12 However, cyclin B1–cdc2 complexes are retained in the cytoplasm on DNA damage, most likely to prevent premature mitosis.28,29 On the other hand, it has been shown that cytoplasmic accumulation of cyclin B1 can induce mitosis by overriding a p53-mediated G2-M checkpoint.30 Thus, cytoplasmic expression of cyclin B1 in colorectal cancer could indicate aberrant cell cycle progression at the G2-M checkpoint, promoting loss of genomic instability and malignant transformation. Moreover, high cyclin B1 expression seems to be an early event in colorectal carcinogenesis; the present study revealed no significant differences in cyclin B1 expression and different pT categories or International Union Against Cancer stages. Our results are in agreement with the findings of Wang et al,21 who studied cyclin B1 expression by Western blot analysis in 41 colorectal carcinomas and demonstrated high expression levels of cyclin B1 in 88% of carcinomas. Concerning the putative prognostic impact of cyclin B1 overexpression, we showed that there were no differences in survival between patients with tumors with high cyclin B1 expression and those with tumors with low expression. This is in contrast with the findings of previous studies on squamous cell carcinomas of the esophagus,17,18 tongue,19 and lung20 that found a poorer prognostic outcome for tumors with high cyclin B1 expression. However, in the latter investigation on lung cancer,20 no prognostic influence was shown in the subgroup of adenocarcinomas, suggesting biologic differences between adenocarcinomas and squamous cell carcinomas. Am J Clin Pathol 2004;122:511-516



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This is the first immunohistochemical study to demonstrate the relationship between cyclin B1 expression and clinicopathologic factors and prognosis in a series of 342 colorectal carcinomas. The present findings show that high cyclin B1 expression is a frequent and an early event during colorectal carcinogenesis; nevertheless, our data suggest that cyclin B1 cannot predict the overall survival of patients with colorectal cancer and its immunohistochemical detection is not a suitable tool for identifying subgroups of patients who might be at higher risk for disease recurrence. The molecular mechanisms leading to high cyclin B1 protein expression in the cytoplasm remain under investigation, and further studies are needed, including analyses of DNA and/or RNA levels and examination of cyclin B1 regulatory proteins. From the 1Department of Histopathology, the Leeds Teaching Hospitals NHS Trust, Leeds, England; Institutes of 2Pathology and 5Statistics, Heinrich-Heine University, Düsseldorf, Germany; 3Department of Surgery, Marien Hospital, Düsseldorf; 4Department of Surgery II, Oita, Japan; and 6Institute of Pathology, Starnberg, Germany. Supported by the Charlotte-and-Alfred-Pierburg-Stiftung, Düsseldorf, Germany. Address reprint requests to Dr Mueller: Gemeinschaftspraxis Pathologie Starnberg, Am Fuchsengraben 3, 82319 Starnberg, Germany. * Drs Grabsch and Lickvers contributed equally to this work. Acknowledgment: We gratefully thank Britta Lemkemeyer for expert technical assistance.

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