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Expression of the Deleted in Colorectal Cancer Gene Is Related to Prognosis in DNA Diploid and Low Proliferative Colorectal Adenocarcinoma

From the Divisions of Oncology and Cell Biology, Department of Biomedicine and Surgery, Linköping University, Linköping, Sweden.

Address reprint requests to Xiao-Feng Sun, MD, PhD, Division of Oncology, Department of Biomedicine and Surgery, Linköping University, S-581 85 Linköping, Sweden; email xisunonk{at}epost.liu.se

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Whether or not the deleted in colorectal cancer (DCC) gene is implicated in metastases or in predicting prognosis in patients with colorectal cancer has not previously been substantiated. Our aims were to investigate DCC expression in primary colorectal cancers and in metastases to identify any prognostic significance.

PATIENTS AND METHODS: DCC expression was examined immunohistochemically in 195 primary colorectal adenocarcinomas and in 23 paired primary tumors and lymph node metastases. DNA content and S-phase fraction were measured by flow cytometry.

RESULTS: The absence of DCC expression was observed in 55 primary tumors (28%). DCC negativity was significantly related to poor prognosis in patients with DNA diploid tumors (P = .03) and those with a low S-phase fraction (< 5%, P = .02) but not in patients with nondiploid tumors or those with a higher S-phase fraction. Furthermore, DCC expression retained its prognostic significance in the diploid subgroup after adjusting for sex, age, site, stage, growth pattern, and differentiation (P = .01). DCC expression was similar in primary tumors and their metastases.

CONCLUSION: The absence of DCC predicted a poor outcome in the patients with diploid tumors and those tumors with a low S-phase fraction. Immunohistochemistry may be considered as a practical test to assess prognosis in this subgroup of patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE DELETED IN colorectal cancer (DCC) gene is located on chromosome 18q21 and is considered a candidate tumor suppressor gene that encodes a transmembrane protein. DCC may have an important functional role in regulating cell proliferation and/or differentiation.¹ The relationship between the DCC gene and colorectal cancer metastases is controversial. Some investigators have reported 18q loss in metastasis,^2-4 which suggests that DCC loss is a late event in tumor development. While others did not confirm this,^5,6 Gotley et al⁶ recently found that the DCC protein detected by Western blotting is not correlated with liver metastases in colorectal cancer patients. The prognostic significance of allelic loss of 18q is not substantiated, although several studies have indicated that patients who lost 18q or lack protein expression in their tumors have an increased death rate.^7-11 These studies highlight the questions of whether the DCC gene is a target of 18q loss of heterozygosity (LOH) and whether DCC expression is a late event in the development of colorectal cancer.

In the present study, we investigated DCC expression in 195 primary colorectal adenocarcinomas and in 23 matched primary tumors and their metastases in the lymph nodes. The aims were to investigate whether the expression of DCC protein was involved in metastasis and whether DCC expression had a potential application in predicting survival in the subgroups of patients. The results indicate that the absence of DCC expression might be an early event, which was related to poor prognosis in the patients with DNA diploid tumors or those tumors with a low of S-phase fraction (SPF).

	PATIENTS AND METHODS

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Patients and Tumor Samples
Blocks of paraffin-embedded tissue were obtained from 195 patients with primary colorectal adenocarcinoma diagnosed at the Department of Pathology, Linköping University, between 1972 and 1986. Among them, there were regional lymph nodes from 23 patients. The patient group consisted of 104 men and 91 women with a mean age 69 years (range, 33 to 93 years). One hundred fifteen tumors were located in the colon and 74 in the rectum. Thirty-one tumors were in Dukes' stage A, 65 were in Dukes' stage B, 64 were in Dukes' stage C, and 32 were in Dukes' stage D. Seventy-two tumors displayed expanding growth, and 91 showed infiltrative growth. One hundred forty-seven tumors were graded as better differentiated and 48 as worse differentiated. Tumor location was unknown in six cases, tumor stage was unknown in three cases, and growth pattern was unknown in 32 cases. None of the patients had received preoperative radiotherapy or chemotherapy. The patients were followed until the end of 1997, and 113 deaths due to the cancer were registered.

Immunohistochemistry
The paraffin-embedded tissue sections were deparaffinized in xylene, rehydrated, and then heated in a 10 mmol/L citric buffer (pH 6.0) for 12 minutes in a 750-W microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), the sections were preincubated with 10% normal rabbit serum to block nonspecific immunostaining. After the blocking solution was removed, monoclonal antibody clone G97-449 (Pharmingen, San Diego, CA) was applied at a dilution of 1:150 for overnight incubation at 4°C. Treatment continued with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dakopatts Co, Glostrup, Denmark) and peroxidase antiperoxidase (PAP) (Dakopatts), with each step lasting for 30 minutes. The slides were washed in PBS between each incubation step. The peroxidase reaction was performed for 8 minutes, using 0.05% 3,3-diaminobenzidine tetrahydrochloride solution (Sigma Chemical Co, St Louis, MO) in PBS which contained 0.003% hydrogen peroxide. The sections were counterstained with hematoxylin for 4 minutes, dehydrated in a series of ethanols, cleared in xylene, and mounted under a coverslip. Sections known to stain positively were used as the positive controls. For negative control, the primary antibody was replaced by the isotype MOPC-141 (Sigma). Seven matched primary tumors and metastases in the lymph nodes were stained twice in the same run of immunostaining in order to avoid bias on the pattern and intensity of staining.

The slides were examined and scored independently by two of the authors without any clinical or pathologic information, with minimal disagreement on scoring. The absence of DCC expression was registered if there were no positive tumor cells. However, we did not regard staining on the margins of sections and areas of poorly presented morphology as specific in order to avoid artifact. The DCC expression was classified as positive if tumor cells were stained, regardless of the intensity of staining (including weak staining). In fact, the staining for DCC on our series was either more than 5% of tumor cells or nothing. In the positive cases, there was a heterogeneity in the intensity of staining and the percentage of stained cells.

Flow Cytometry
Fifty-micrometer sections from the same paraffin-embedded block used for the immunostaining were taken for flow cytometry. DNA diploid tumors had a single G₀/1 cell cycle peak at the same level as the internal control cells, and nondiploid tumors had an additional distinct G₀/1 cell cycle peak. SPF was estimated by using a rectangular model. The details were described previously.¹²

Statistical Analysis
The ² method was used to test the association of DCC expression with other variables.¹³ Cox's proportional hazards model was used to estimate and test the prognostic significance.¹⁴ The curves describing survival were computed according to the method of Kaplan and Meier.¹⁵ All P values cited are two-sided, and P values less than 5% were judged as statistically significant.

	RESULTS

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Relationship Between DCC Expression and Clinicopathologic and Other Factors
Among 195 tumors, 55 (28%) exhibited a negative immunohistochemical reaction for DCC (Fig 1A) and 140 (72%) were DCC-positive in the cytoplasm (Fig 1B). Table 1 summarizes the relationships of DCC expression with clinicopathologic variables. We did not find significant relationships between DCC expression and patients' sex, age, tumor location, Dukes' stage, growth pattern, or grade of differentiation (P > .05).

View larger version (136K): [in this window] [in a new window]   Fig 1. Immunohistochemical analysis of the DCC protein by monoclonal antibody G97-440 in colorectal adenocarcinoma. The sections received a light hematoxylin counterstain. Panel A shows the absence of DCC protein, and panel B shows positive DCC expression.

Among 184 cases available for DNA measurement, 87 (47%) were DNA diploid and 97 (53%) were nondiploid. Excluding 37 cases in which SPF could not be estimated, 40 tumors (27%) had an SPF of less than 5%, 51 (34%) had an SPF of 5% to 10%, and 56 (38%) had an SPF of 10%. DCC expression was not associated with DNA ploidy (P = .34), but the absence of DCC expression was related to lower SPF (P = .05, Table 1).

DCC Expression in Primary Tumors and Metastases
In 23 cases, both primary tumors and their lymph node metastases were available for comparison of DCC expression. All metastases in the lymph nodes showed the same staining pattern as seen in the primary tumors (four negative and 19 positive cases), although the intensity of staining varied in a few paired samples.

DCC Expression in Relation to Survival
In univariate survival analysis, the negative expression of DCC predicted a worse prognosis for patients with DNA diploid tumors (P = .03, Fig 2A) and patients with a low SPF (< 5%; P = .02, Fig 3A), but not for patients with nondiploid tumors (P = .41, Fig 2B) and those with a higher SPF ( 5%; P = .25, Fig 3B). Furthermore, DCC expression was an independent prognostic factor after adjustment for patients' sex, age, tumor location, Dukes' stage, growth pattern, and grade of differentiation (P = .008, Table 2). We did not perform multivariate analysis for patients with an SPF of less than 5% because of the small number of cases.

View larger version (12K): [in this window] [in a new window]   Fig 2. The expression of DCC in relation to prognosis in the patients with DNA diploid tumor (A) and those with nondiploid tumor (B).

View larger version (12K): [in this window] [in a new window]   Fig 3. The expression of DCC in relation to prognosis in the patients with less than 5% of SPF tumor (A) and those with 5% SPF tumor (B).

Multivariate analyses demonstrated that the association with prognosis of DCC expression differed significantly between the patients with DNA diploid tumors and those with nondiploid tumors (P = .03). The association also differed significantly between the patients with less than 5% SPF tumors and those with 5% or more SPF tumors (P = .01). DCC expression was not related to prognosis in all patients or in the patients with individual Dukes' stage tumor (P > .05).

	DISCUSSION

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It has been reported that LOH at the locus on 18q is responsible for the absence of DCC expression into mRNA in colorectal tumors.¹⁶ Recently, Shibata et al⁹ indicated that the absence of DCC expression, as determined by immunohistochemistry, is an adverse prognostic factor in the patients with stage II or III colorectal cancer. In this study, we found that the lack of DCC expression predicted an unfavorable outcome in a subgroup of patients. Whether the absence of DCC expression was the only important result of the LOH in the DCC gene is unclear. At least two other candidate tumor-suppressor genes, VJ18-1 (18q21.1) and SMAD4 (18q21.1), are located close to the DCC gene (18q21.3).^1,17-20 The region lost may contain these two genes in addition to the DCC gene, and potentially any of these genes may affect prognosis. However, some studies indicated that deletion and mutation of JV18-1 and SMAD4 are not common in colorectal cancer,^18,21-23 suggesting a limited role as compared with that of the DCC gene in colorectal cancer development. Moreover, there might be another tumor suppressor gene(s) in this region that is involved in tumor development.

Ookawa et al⁵ reported that 18q LOH is more frequent in liver metastases than in primary tumors, on the basis of a comparison of unmatched primary tumors with metastases. However, they further observed that in 13 cases of both primary tumors and liver metastases from the same patients, LOH of 18q was detected in all samples. Shortly thereafter, Iino et al³ evaluated 18q LOH in three cases of liver metastasis and three cases of lymph node metastasis and indicated a similar pattern of 18q LOH in the metastases as in the primary tumors. Furthermore, Gotley et al⁶ revealed that DCC protein detected by Western blotting is present in both colorectal cancer and liver metastases. Here, we studied DCC expression in both primary tumors and lymph node metastases from the same patients and found no difference in staining pattern in matched primary tumors and metastases, although considerable care was taken to stain matched cases in the same run of immunostaining. In accordance with the findings by others,^9,24 we did not find that DCC expression was related to tumor stage. Taken together, these results suggest that DCC may not be a critical prerequisite for tumor cells to progress, or to acquire metastatic ability, which argues against DCC expression being a late event in the development of colorectal cancer.

In the present study, we found that the absence of DCC expression predicted poor survival in the patients with DNA diploid tumor or those with lower SPF tumor, but DCC expression was not significantly related to prognosis in the patients with nondiploid tumor nor in patients with an increased SPF. Further multivariate interaction analyses showed that the association with prognosis of DCC expression differed significantly between the two DNA groups (P = .03), as well as between the two SPF groups (P = .01). In addition, DCC expression clearly had a prognostic value independent of patients' sex, age, tumor location, Dukes' stage, growth pattern, and differentiation (P = .008). It has been suggested that the evolutionary pathway in the tumor is via a change from a DNA diploid to a tetraploid, by doubling the chromosome number, followed by a loss or gain of chromosomes, producing aneuploidy. Meanwhile, a low S-phase in the tumor evolves to a high S-phase.^25,26 Our earlier study on the same series demonstrated that both tetraploid and aneuploid tumors had a high SPF compared with diploid tumors.¹² Furthermore, p53 expression, as a late event, tended to be higher in DNA nondiploid tumors than in diploid tumors.²⁷ Therefore, we believe that DNA diploidy and low proliferation are indicators of early-stage tumor development. The studies by Jen et al⁷ and Martinez-Lopez et al¹¹ demonstrated that the allele loss of 18q predicts a poor outcome in patients with tumor-node-metastasis stage II colorectal tumors^7,11 but not in patients with stage III tumors.⁷ Our findings, in association with their results, suggest that the absence of DCC expression predicts poor prognosis in the earlier stages of colorectal cancer.

We did not find that DCC expression predicted survival in individual Dukes' stages. The number of cases in each stage is small, but this is in accordance with the results of O'Connell et al.²⁸ Their results failed to reach statistical significance on the prognostic value of 18q loss in the patients with Dukes' stage B or C, although there was a trend. Others have reported that in stage II^7,9,11 or III^9,11 classified using the tumor-node-metastasis system, the loss of 18q or the absence of DCC protein is a prognostic factor. The reason for this discrepancy is unclear, but it may depend on limited numbers or on different classifications of tumor stage.

Compared with the frequency of absent DCC expression (50%) observed by Shibata et al,⁹ we had a lower rate of negative expression (28%). The main reason for this discrepancy may be the different antibodies used and the criteria used for determining positive and negative DCC reactions. Shibata et al used a panel of antibodies that included monoclonal antibody G97-447, which is the same one we used, and three polyclonal antibodies (721,723, and 724). They regarded cases as positive when at least 25% of tumor cells were stained, but they did not state the intensity of staining. They observed an "all-or-nothing" phenomenon for the DCC staining. In the present study, the DCC expression was classified as a positive if any tumor cells were positive, regardless of the intensity of staining. By using a monoclonal antibody, we did not see an all-or-nothing phenomenon. Indeed, the DCC antibody tended to stain at least 5% of tumor cells if it was the tumor was positive. However, the percentage and the intensity of staining varied among positive cases.

In conclusion, our data suggests that the absence of DCC protein predicted a survival disadvantage in the patients with DNA diploid or slowly replicating colorectal tumors. This supports evidence that the DCC gene may be a target of 18q LOH in colorectal cancer. Immunohistochemistry may be considered as a practical test to assess prognosis in this subgroup of patients.

	ACKNOWLEDGMENTS

 
Supported by grants from the Swedish Cancer Society, The Östergötland County Research Fund, and the Linköping Medical Society.

We thank Dr Douglas Rogers for the linguistic revision.

	REFERENCES

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Submitted October 6, 1998; accepted February 18, 1999.

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