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Annals of Surgical Oncology 8:533-537 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Bcl-2 Is a Useful Prognostic Marker in Dukes’ B Colon Cancer

Sarkis H. Meterissian, MD, Maria Kontogiannea, BSc, Mohamed Al-Sowaidi, MD, Ayman Linjawi, MD, Fawaz Halwani, MD, Bruce Jamison, MD and Michael Edwardes, PhD

From the Departments of Surgery (SHM, MK, MA-S, AL), Pathology (FH, BJ), and Biostatistics (ME), Royal Victoria Hospital, Montreal, Quebec.

Correspondence: Address correspondence to: Dr. S. Meterissian, Royal Victoria Hospital, 687 Pine Avenue West S10.22, Montreal, Quebec H3A lA1; Fax: 514-843-1503.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Currently, the use of adjuvant therapy specifically in Dukes’ B colon cancers is controversial, emphasizing the importance of identifying prognostic markers to select patients for such therapy. Bcl-2 plays an important role in apoptosis regulation of solid tumors, such as colon and breast cancer, and is normally expressed in the base of the colonic crypts. The purpose of this study is to determine whether or not bcl-2 expression can be used to predict survival in Dukes’ B colon cancer patients.

Methods: Charts of 76 patients operated on at the Royal Victoria Hospital from 1986 to 1992 were reviewed. Bcl-2 staining was done with the avidin-biotin-peroxidase complex method using commercially available monoclonal bcl-2 antibodies. Two pathologists graded the intensity of bcl-2 staining on a scale of 0–3 and estimated the percentage of tumor cells staining positively (T-percent). Univariate and multiple regression of factors on overall survival (OS) and disease-free survival (DFS) was done with a Cox proportional hazards model and Kaplan-Meier survival curves.

Results: The mean age was 71.2 years, with 41 female and 35 male patients. Mean tumor size was 5.4 cm with tumor grades of 19 well, 52 moderate, and 5 poorly differentiated. Tumors expressing bcl-2 had a similar DFS (P = .14) but a significantly improved OS (P = .04) compared with the bcl-2 negative tumors. The risk ratio for DFS was 0.49 (95% CI, 0.19–1.26) and for OS was 0.35 (95% CI, 0.13–0.94).

Conclusions: These data indicate that enhanced bcl-2 expression, specifically in Dukes’ B colon carcinomas, is associated with improved survival. Thus, patients whose tumors do not express bcl-2 should be considered for adjuvant therapy.

Key Words: Apoptosis • Colon cancer • Prognosis • Bcl-2 • Dukes’ B


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer is a major cause of cancer-related morbidity and mortality in North America.1 Presently, the primary treatment for colorectal cancer is surgical resection with adjuvant therapy reserved for Dukes’ C colon and Dukes’ B and C rectal carcinoma. The drug combination of 5-fluorouracil (5-FU) plus levamisole, given for 1 year, was shown to be unequivocally associated with improved survival2,3 in Dukes’ C colon cancer. Even though chemotherapy has not been shown to be effective in Dukes’ B colon cancer, use of specific prognostic markers may allow identification of a subset of patients with a poor predicted outcome who may then benefit from adjuvant chemotherapy.

Bcl-2 (B-cell lymphoma/leukemia-2) was initially identified at a breakpoint in a chromosomal translocation (t14:18) that occurs in human B-cell lymphomas. Bcl-2 is a known inhibitor of apoptosis4,5 and has also been shown to block chemotherapy-induced apoptosis6 and p53-induced apoptosis7 in some hemopoietic cell lines. The ability of bcl-2 to inhibit apoptosis depends on the intracellular balance among a number of its family members, including BAG1, Bad, Bax, bcl-xL and bcl-xS. Bcl-xL, like bcl-2, functions as an inhibitor of apoptosis, whereas bcl-xS serves as a dominant negative inhibitor of bcl-xL and bcl-2.8 The bcl-2 binding protein, BAG1, inhibits apoptosis9 whereas the Bad and Bax proteins promote apoptosis.10,11 Expression of bcl-2 has been studied in a number of human tumors, including colorectal carcinoma. Non-small-cell lung carcinomas,12 breast carcinomas,13,14 and neuroblastomas15 all express bcl-2, and studies have been published correlating expression with survival. Similarly, a number of studies16–20 have been published evaluating the clinical use of bcl-2 immunohistochemical expression as a prognostic factor. But only one of these studies has looked specifically at Dukes’ B20 colon carcinoma, which is associated with an up to 20% to 30% chance21 of recurrence and death. The identification of sensitive prognostic markers in this group of patients would allow the use of postoperative adjuvant therapy in a subset of patients with a worse prognosis, with a resultant improvement in survival. Therefore the purpose of this study was to evaluate, using immunohistochemical analysis, the prognostic value of bcl-2 expression specifically in Dukes’ B colon cancers not previously treated with adjuvant therapy.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Patients with Dukes’ B colon carcinoma operated on between 1986 and 1992 were identified from the Tumor Registry of the Royal Victoria Hospital. Patients with rectal carcinoma, those who died within 30 days of surgery, and those who received adjuvant chemotherapy and/or radiotherapy were excluded, leaving a total of 76 patients for this study. The clinicopathologic factors studied included age, sex, tumor size, and tumor grade. Tumor tissues, which were formalin-fixed and paraffin-embedded, were obtained from the Pathology Department of the Royal Victoria Hospital. For each patient, one paraffin block was selected based on good morphological preservation and the presence of adjacent normal mucosa to act as an internal control (because the lymphocytes in the lamina propria stain positively for bcl-2). This study was approved by the Institutional Review Board of the Department of Surgery.

Staining Procedure
Paraffin-embedded, formalin-fixed tissues were sectioned at 3 µm and placed on poly-L-lysine covered slides. After overnight incubation at 37°C, the tissues were dewaxed and rehydrated with sequential washes in xylene and ethanol. To improve staining, slides were microwaved for 30 minutes at 65°C in 10-µM citrate buffer (pH 6.0) for antigen retrieval. After cooling to room temperature, slides were treated for 30 minutes in 0.3% hydrogen peroxide diluted in methanol to inhibit endogenous peroxidase activity. After extensive washing, the slides were sequentially incubated with (1) 2% normal goat serum and 2% bovine serum albumin diluted in 0.1M Tris (pH 7.6) for 1 hour at room temperature; (2) 1:50 dilution of bcl-2 antibody (mouse antihuman, DAKO Corporation, Carpenteria, CA) in Tris buffer and 0.5% bovine serum albumin (0.5% BSA) overnight at 4°C; (3) 1:100 dilution of goat antimouse biotinylated antiserum (Jackson Immuno Research, West Grove, PA) in 0.1M Tris (pH = 7.6) with 0.5% normal human serum and 0.5% BSA for 1 hour at room temperature; and (4) ABC-horseradish peroxidase (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes at room temperature. The enzyme reaction was developed using diaminobenzidine (DAB) with hematoxylin used as a counterstain. The slides were mounted with Permount. As described above, lymphocytes within the intestinal mucosa served as an internal positive control for bcl-2 immunoreactivity, and negative controls were obtained by omission of the primary antibody.

Semiquantitative Assessment of Immunohistochemical Staining Pattern
Bcl-2 immunostaining was evaluated by two pathologists who were blinded to the clinical outcome. The staining was categorized as either positive or negative based on (1) intensity of the immunoreactivity (scale of 0–3 with 0 = negative, 1 = faint, 2 = moderate or light brown, and 3 = dark brown) and (2) percentage of tumor cells staining positively (T-percent). A tumor was deemed negative for bcl-2 if the following two criteria were realized: staining intensity scale <=1 and T-percent <=30%.

Follow-up Data
Patient outcome was determined from the hospital and physicians’ office charts. Mean follow-up was 59 months. Disease-free survival (DFS) was calculated from the date of surgery to the date of first recurrence whereas overall survival (OS) was calculated from surgery to death from disease.

Statistical Analysis
Univariate and multiple regression of factors on OS and DFS were done with Cox proportional hazards model,22 which estimates a hazard ratio with a P value (two-sided) and 95% confidence interval (CI) for each factor. The hazard ratio for a factor with two categories (such as bcl-2) is the rate ratio of the two rates of event (disease or death), such as positive bcl-2 rate over negative bcl-2 rate. Kaplan-Meier survival curves22 estimate the proportion that would survive up to a given time, assuming no competing causes of death. All computations and figures were done with EGRET (Pecan Version 1.02.10; Cytel Software Corporation, Cambridge, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient and Tumor Characteristics
These are summarized in Table 1. Of the 76 patients, 18 were dead of disease and 8 from other causes after a mean follow-up of 59 months. The tumors were fairly large at 5.4 cm, with most classified as moderately differentiated.


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  		TABLE 1. Patient and tumor characteristics
 
Pattern of Bcl-2 Staining
Of the 76 paraffin-embedded formalin-fixed tissues of colorectal cancer, 14 (18%) did not demonstrate bcl-2 staining (with 3 completely negative). The bcl-2 cytoplasmic staining in the adjacent normal tissue was found in the basal area of the crypts, as expected. In all cases, lymphocytes in the lamina propria stained positively, serving as an efficient internal positive control. Bcl-2 staining of the tumors varied as follows, based on tumor intensity (TI) measurements: 3 patients had a TI of 0, 27 patients had a TI of 1, 31 patients had a TI of 2, whereas 15 patients had the maximal staining of 3 (Fig. 1). Bcl-2 staining did not vary with tumor grade (Table 2) although the number of patients with poorly differentiated tumors was low.



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  		FIG. 1. Characteristic cytoplasmic bcl-2 staining in a colon adenocarcinoma. Note the positive staining of lymphocytes in the lamina propria.

 

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  		TABLE 2. Effect of tumor grade on Bcl-2 staining
 
Correlation of Bcl-2 Staining and DFS and OS
The OS (time from surgery to disease-related death) was 60.4 (range, 5–110) months. The average follow-up time (i.e., time to death or to study end) was 59 (range, 5–110) months. P values and 95% confidence intervals are denoted P and CI, respectively. By univariate analysis, tumor grade (P = .522), tumor size (P = .054), and bcl-2 staining (P = .140; rate ratio = .49, 95% CI, .19–1.26) were not strong predictors of DFS. Both bcl-2 staining and tumor size showed a stronger association with OS (P = .048 and P = .045, respectively) with the bcl-2 rate ratio = .35 (95% CI, .13–.94) and the rate ratio per cm tumor size = .72 (95% CI, .53–.99). In a bivariate regression of bcl-2 and tumor size, adjusted rate ratios were about the same and both P values were between .05 and .20, so the effects of tumor size and bcl-2 on survival are almost independent of each other. Figures 2 and 3 depict Kaplan-Meier survival curves for bcl-2 negative and positive stains, to which the Cox model (or log-rank test) P values .140 and .038, respectively, apply to the test curve difference. As can be seen, patients with tumors staining positively for bcl-2 had a statistically significant improvement in survival.



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  		FIG. 2. Kaplan-Meier curve for disease-free survival of tumors of varying bcl-2 expression. The tics represent censored patients (P = .140)

 


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  		FIG. 3. Kaplan-Meier curve for overall survival of tumors of varying bcl-2 expression. The tics represent censored patients (P = 0.038)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Identification of molecular markers predicting prognosis in Dukes’ B colon carcinoma can allow selection of patients for adjuvant therapy. This study demonstrates that tumors lacking bcl-2 expression have a significantly worse prognosis. Bcl-2 is an inhibitor of apoptosis and, as noted in our study, it should therefore be expressed normally in the basal cells of the colonic crypts. Its expression is gradually lost as one progresses up the crypt to the tip where cells are shed via a mechanism of apoptosis. Expression of bcl-2 in areas other than the colonic crypts would therefore be considered abnormal and, as seen here, almost all the tumors, regardless of differentiation, expressed varying levels of this protein. Others have also looked at the relationship between bcl-2 expression and survival with varying results. Hague and colleagues23 found increased bcl-2 expression in 19 of 22 adenocarcinomas and in 12 of 13 adenomas. Bhatavdekar et al.18 found that tumors positive for bcl-2 had a worse prognosis, with a direct correlation between tumor grade and expression, with poorly differentiated tumors being low expressors. However, Sinicrope et al.,20 Baretton et al.,17 and Ofner et al.18 showed that colorectal carcinomas expressing bcl-2 have a favorable prognosis, although none of these studies specifically looked at Dukes’ B colon carcinomas.

A similar relationship between bcl-2 expression and a favorable outcome has been noted in breast cancer. Gasparini et al.24 analyzed 180 cases of primary node positive carcinomas of the breast for bcl-2 protein. Paradoxically, as in our study, they found that the 5-year survival was significantly better among patients with bcl-2 positive tumors. The expression of bcl-2 in breast cancer has also been found to be a significant predictor of estrogen receptor positivity.25 Similarly Pezzella et al.12 demonstrated that bcl-2 positive non–small-cell carcinomas of the lung had a statistically higher OS.

The mechanisms for the paradoxical relationship noted in our study between bcl-2 expression and survival is unknown. Some of the theories include the observation that bcl-2 in several solid tumor cell lines resulted in a paradoxical growth inhibition similar to that seen with wild-type p53.26 This growth inhibition or retardation may decrease the rate of acquisition of complementary genetic defects that would otherwise increase the metastatic potential of the tumor cells. Another explanation for our results could be the presence or absence of other members of the bcl-2 family that may modulate bcl-2 function27 such as Bax. Bax is an important regulator of bcl-2 mediated apoptosis, as the overall regulation of apoptotic stimuli by the ratio of Bax to bcl-2 has been documented.10 Thus, tumors with elevated bcl-2 expression may also be high Bax expressors, thus explaining the favorable outcome. Loss of Bax expression may be seen in tumors with decreased bcl-2 expression, which may then lead to metastatic disease and death. Such a model has already been described in breast cancer28 and, thus, is quite plausible.

In summary, our study demonstrates that bcl-2 expression correlates with improved survival, specifically in Dukes’ B colon carcinoma, and this can be used as a prognostic marker. Immunohistochemical staining for bcl-2 in this subgroup of patients can help in the selection of a high-risk group who would theoretically benefit from adjuvant therapy. Studies are presently underway in our laboratory looking at other members of the bcl-2 family, in addition to looking at whether or not tumors expressing bcl-2 are more chemoresponsive.


   Acknowledgments
 
This study was done at the Royal Victoria Hospital and supported by a grant from the Cedars Cancer Foundation. The authors thank Ms. Ennia Mulfati for secretarial assistance.


   Footnotes
 
Presented at the 51st Annual Meeting of the Society of Surgical Oncology, San Diego, California, March 26-29, 1998.

Received for publication November 21, 2000. Accepted for publication February 28, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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