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Prognostic Significance of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand and Its Receptors in Adjuvantly Treated Stage III Colon Cancer Patients

Caroline M. van Geelen, Jantine L. Westra, Elisabeth G. de Vries, Wytske Boersma-van Ek, Nynke Zwart, Harry Hollema, H. Marike Boezen, Nanno H. Mulder, John T. Plukker, Steven de Jong, Jan H. Kleibeuker, Jan J. Koornstra

From the Departments of Medical Oncology, Medical Genetics, Gastroenterology and Hepatology, Pathology, Epidemiology, Surgery, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands

Address reprint requests to Jan J. Koornstra, MD, Department of Gastroenterology and Hepatology, University Medical Center, University of Groningen, PO Box 30001, 9700 RB Groningen, the Netherlands; e-mail: j.j.koornstra{at}int.umcg.nl

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions REFERENCES

 
Purpose: In preclinical models, there is synergism between chemotherapy and recombinant human tumor necrosis factor (TNF) –related apoptosis-inducing ligand (TRAIL) on apoptosis induction in tumor cells. Therefore, the prognostic relevance was analyzed of the expression of TRAIL and its death receptors DR4 and DR5 on disease-free survival and overall survival in stage III colon cancer patients treated with adjuvant chemotherapy.

Methods: Tissue microarrays were constructed of primary tumor tissue from 376 stage III colon cancer patients treated in a randomized adjuvant chemotherapy study (fluorouracil/levamisole v fluorouracil/levamisole/leucovorin) and stained immunohistochemically for TRAIL, DR4, and DR5. Log-rank tests and Cox proportional hazard analysis, with adjustment for treatment arm, sex, age, N stage, microsatellite instability status, and p53 mutation status, were performed.

Results: The majority of tumors showed high expression of TRAIL (83%), DR4 (92%), and DR5 (87%). Median follow-up was 43 months. High DR4 expression was associated with worse disease-free survival (odds ratio [OR] = 2.19; 95% CI, 1.06 to 4.53; P = .03), worse overall survival (OR = 2.22; 95% CI,1.03 to 4.81; P = .04) and shorter time to recurrence (P = .02) compared with those with low DR4 expression. TRAIL or DR5 expression had no prognostic value.

Conclusion: High DR4 expression is associated with worse disease-free and overall survival in stage III adjuvant-treated colon cancer patients. Evaluation of DR4 expression in stage III colon cancer patients may identify a subset requiring more aggressive adjuvant treatment.

	INTRODUCTION

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Colorectal cancer is one of the leading causes of cancer-related death in the Western world. Of patients who undergo potentially curative surgery alone, up to 50% patients ultimately relapse and die of metastatic disease.¹ Adjuvant treatment aims to eliminate residual tumor cells and increase the proportion of patients achieving long-term disease-free and overall survival. In patients with established locoregional lymph node metastasis (stage III), adjuvant fluorouracil-based chemotherapy after surgical resection improves survival. However, intrinsic and acquired resistance to chemotherapeutic drugs remain a major problem. It is therefore important to identify prognostic factors that can help to develop new patient-tailored treatment strategies for colorectal cancer patients.

The tumor suppressor gene p53 plays a key role in induction of the intrinsic apoptosis pathway in response to several chemotherapeutic agents. Most colorectal cancers have inactivated p53 and this may limit the efficacy of chemotherapy.^2-4 Data from our institution recently confirmed the importance of p53 mutations as a prognostic factor in adjuvantly treated stage III colon cancer patients.⁵ Tumor necrosis factor (TNF) –related apoptosis-inducing ligand (TRAIL) is a so-called death ligand of the TNF family that induces apoptosis in tumor cells.^6,7 Interestingly, in contrast to most chemotherapeutic agents, recombinant human (rh) TRAIL can initiate apoptosis through a distinct (extrinsic) apoptosis pathway, independent of the p53 status of the tumor.⁴ Furthermore, chemotherapeutic agents cooperate with rhTRAIL and facilitate p53-independent apoptosis of colon cancer cells.⁴ rhTRAIL, alone or in combination with chemotherapy, has demonstrated an antitumor effect in preclinical studies.^8-13 It therefore harbors potential as a cancer therapeutic agent.

TRAIL binds to four membrane receptors.^14-16 After binding to the death receptors DR4 and DR5, TRAIL triggers apoptosis in tumor cells. The other two receptors, decoy receptors 1 and 2 (DcR1 and DcR2) do not transfer an apoptosis signal because they either lack an intracellular domain or have a truncated intracellular domain, respectively. Expression patterns of TRAIL, DR4, and DR5 have already been described in normal colon, sporadic adenomas, and sporadic carcinomas.^17,18 Recently, DR5 expression was shown to be important for sensitivity to fluorouracil.¹⁹ Sträter et al found that DR4 expression was a prognostic factor for colorectal cancer.¹⁸ Their study included both stage II and III patients. No robust data are available on TRAIL, DR4, and DR5 expression in tumors of patients who receive adjuvant chemotherapy for stage III colon cancer. This would be of major interest because of the observed synergism between chemotherapy and rhTRAIL on apoptosis induction in tumor cells and the potential clinical application of rhTRAIL. Recent reports have indicated that besides apoptotic signals, other, antiapoptotic, signaling pathways can be activated by TRAIL or its death receptors that may provide tumors with a growth advantage.^20-22 To allow further clarification of the role of TRAIL and its death receptors in colon cancer, we undertook this study.

The aim was to determine expression patterns of TRAIL, DR4, and DR5 and to correlate these with disease-free and overall survival using tissue microarrays of 376 primary tumors of stage III colon cancer patients who participated in an adjuvant chemotherapy study.

	METHODS

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Patients' Material
Formalin-fixed paraffin-embedded primary tumor tissue from 376 of the 500 patients with stage III colon cancer who were included in a nationwide randomized trial was available. The remaining 124 patients, from whom no primary tumor specimens or insufficient material were available, did not differ from the study population with respect to baseline characteristics as age, sex, tumor location or outcome. All patients had undergone surgical resection with histologically negative resection margins and were treated adjuvantly with fluorouracil-based chemotherapy (fluorouracil/levamisole or fluorouracil/levamisole/leucovorin as described earlier).²³ The medical ethical committees of all participating hospitals approved the study. All patients gave written informed consent.

Clinical and Tumor Characteristics
Clinical and tumor characteristics were derived from the clinical database maintained at the Comprehensive Cancer Center North Netherlands (Groningen, the Netherlands). The mean age of the 376 patients at random assignment was 58.8 years (range, 26.2 to 75.8 years), median age was 60.0 years, 201 (53.5%) were males. One hundred seventy-three tumors (46%) were right-sided. By histologic classification according to WHO criteria,²⁴ the degree of differentiation was good in 16.2%, moderate in 53.5%, poor in 22.6% and unknown in 7.7%. With respect to lymph node status, 74% of the resection specimens had N1 nodal involvement (one to three positive regional nodes), while 26% concerned N2 disease ( four regional nodes).²⁵ Microsatellite instability (MSI) had been determined previously.⁵ MSI data were available for 263 of 376 tumors. High-frequency MSI (MSI-H) was found in 44 (16.7%) of 263 cases. The 376 patients were equally distributed between treatment with fluorouracil/levamisole (50.8%) and fluorouracil/leucovorin/levamisole (49.2%). The addition of leucovorin had no effect on disease-free or overall survival.²³ During a mean follow-up of 43 months, 160 deaths were observed. Median follow-up time of all 376 patients was 43.5 months (range, 2.6 to 130.4 months) and for those alive at the end of follow-up (n = 216) it was 62.5 months (range, 4.9 to 130.4 months).

Tissue Microarrays
The construction of tissue microarrays from this patient group was recently described.²⁶ Briefly, representative tumor tissue samples were selected using hematoxylin and eosin–stained slides and arrayed into four different tissue microarrays. Three 0.6-mm tissue cores were taken from three discrete representative regions of each tumor specimen using the manual tissue arrayer from Beecher Instruments (Sun Prairie, WI) and put into a standard-size recipient paraffin tissue microarray block. Each array contained 322 tissue cores, representing 94 tumor samples in triplicate and 20 internal controls (10 nontumor and 10 tumor tissue) in duplicate on all arrays. Sections of 4 µm were cut from each array block using the paraffin tape-transfer system with adhesive-coated slides.

Immunohistochemistry
Tissue microarrays were stained for TRAIL, DR4, and DR5. After deparaffinization in xylene, blocking of endogenous peroxidase with 3% hydrogen peroxide and incubation with avidin and biotin blocking solutions (Vector Laboratories, Burlingame, CA), the primary antibodies were applied for 1 hour at room temperature. Staining procedures for TRAIL, DR4, and DR5 were performed as described previously.¹⁷ Briefly, slides were stained with a goat polyclonal immunoglobulin G (IgG) for TRAIL (1:25; clone K-18, Santa Cruz Biotechnology, Santa Cruz, CA), a goat polyclonal IgG for DR4 (1:50; clone C-20, Santa Cruz Biotechnology) and a rabbit polyclonal IgG for DR5 (1:100; Oncogene Research, Cambridge, MA). After washing with phosphate-buffered saline (PBS), slides were incubated with a 1:300 dilution of a biotinylated rabbit-antigoat or swine-antirabbit antibody (DAKO, Glostrup, Denmark) respectively, followed by addition of streptavidin-conjugated peroxidase. Peroxidase activity was visualized with diaminobenzidine. Slides were counterstained with hematoxylin. Evaluation of staining was performed by light microscopy by two investigators, without knowledge of the clinical data and with re-evaluation under a multiheaded microscope if results did not agree. Cores containing less than 10% tumor cells were not evaluated. If cores from one tumor differed in staining intensity, the median score of the three related cores determined the ultimate score. Immunoreactivity was assessed using a grading system of four categories based on expression intensity (0 = no, 1 = low, 2 = moderate, 3 = strong). For statistical purposes, categories 0 and 1 were considered as low and categories 2 and 3 as high expression.

Statistical Analyses
Disease-free survival with a follow-up of at least 3 years is increasingly being considered an appropriate end point in studies evaluating treatment effects in adjuvant treated colon cancer patients, preferable over overall survival.²⁷ With the increasing availability of newer agents and regimens, the expected median survival after recurrence will be longer. Thus, overall survival is less likely to predict the value of the initial treatment as a prognostic marker. Disease-free survival was defined as the period from date of random assignment to date of any documented relapse or date of last contact without any documented relapse. Overall survival was defined as the time period from randomization to documented death or last contact and documented alive. As a third end point, a surrogate marker for tumor biology, we analyzed time to recurrence, defined as the interval between random assignment and documented tumor recurrence.

Differences in distributions of categoric variables between groups were tested using ² tests. Differences between groups in distributions of continuous variables were tested using t tests or Mann-Whitney U tests, as appropriate. Patients were categorized into low and high expression levels of TRAIL, DR4, and DR5. Differences in disease-free and overall survival according to expression of TRAIL, DR4, and DR5 were analyzed using log-rank testing and Cox proportional hazard analysis. P values less than .05 were considered significant. SPSS for Windows 12.0 (SPSS Inc, Chicago, IL) was used for all statistical analysis.

	RESULTS

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Expression of TRAIL, DR4, and DR5
Immunohistochemistry for TRAIL, DR4, and DR5 was performed on all 376 tumors. Interpretable data were obtained for TRAIL in 313, for DR4 in 315, and for DR5 in 341 tumors. TRAIL, DR4, and DR5 staining was cytoplasmic and no apparent membranous staining was detected. High expression (staining intensity 2 and 3) was observed in 83% of the tumors for TRAIL, 92% for DR4, and 87% for DR5, respectively (Table 1; Fig 1). When coupling interpretable staining intensity data of DR4 and DR5 expression, eight of 291 tumors showed low expression of both receptors; in all other cases expression of at least one death receptor was high. DR4 and DR5 expression was similar in tumors with high TRAIL expression and low TRAIL expression. Staining intensities of TRAIL and TRAIL receptors were not correlated with histologic grade.

View larger version (117K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. DR4, DR5, and TRAIL expression in tissue microarrays of colon cancer tumors. Examples of immunohistochemical staining results for (A and B, low and high expression, respectively) DR4; (C and D, low and high expression, respectively) DR5; and (E and F, low and high expression, respectively) TRAIL. DR, death receptor; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

View larger version (9K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Disease-free survival in adjuvant-treated stage III colon cancer patients according to (A) TRAIL, (B) DR4, and (C) DR5 expression. DR, death receptor; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

Time to Recurrence in Relation to TRAIL, DR4, and DR5 Expression
Of the 376 patients, 168 (44.7%) experienced tumor recurrence. High DR4 expression was associated with a shorter time to recurrence compared with low DR4 expression (mean, 16.6 v 18.2 months; median, 11.4 v 19.9 months; P = .02). Time to recurrence was comparable between tumors with low or high expression of DR5, and TRAIL respectively.

Multivariable Analysis of Prognostic Factors
DR4 and DR5 expression are at least in part p53 dependent.^28,29 Therefore, expression patterns of DR4 and DR5 were compared between tumors with p53 mutations and those with wild-type p53. No significant differences were found between the two groups. Analysis of other potential prognostic variables as age, sex, treatment arm, tumor grade, T stage, N stage, MSI status, and p53 mutation status revealed that N1 stage, MSI-H, and wild-type p53 were independent favorable prognostic factors for disease-free and overall survival.⁵ We therefore repeated all Cox proportional hazard analyses to estimate the effect of TRAIL, DR4, and DR5 expression on disease-free and overall survival with additional adjustment for N stage, MSI status and p53 mutation status. This did not change the associations described herein.

	DISCUSSION

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In the present study, the prognostic value of TRAIL and its death receptors DR4 and DR5 was studied in tumors of 376 stage III colon cancer patients who received adjuvant chemotherapy. The majority of the tumors showed high expression of TRAIL, DR4, and DR5. High DR4 expression was associated with worse disease-free and overall survival and a shorter time to recurrence. There was a trend toward better overall survival with high DR5 expression as compared with low DR5 expression.

In a similar study, high DR4 expression was associated with better disease-free survival rates, which is in contrast to our results.¹⁸ That study was performed in a heterogeneous group of 128 colon cancer patients, including both stage II and III disease, of whom only 18.6% received adjuvant chemotherapy.¹⁸ The strengths of our study are the high number of patients, the inclusion of only stage III colon cancer patients and the fact that all patients received fluorouracil-based chemotherapy within a prospective trial. This means that results obtained give a level II evidence for the utility of the tumor marker studied.³⁰

In our study, high DR4 expression was associated with worse disease-free and overall survival. This suggests that stage III colon cancers with high DR4 expression may have a growth advantage rather than a pro-apoptotic phenotype. This may seem unexpected, because high DR4 expression suggests apoptotic susceptibility. Recent studies, however, demonstrated that in several tumor cell lines, including a colon cancer cell line, TRAIL can mediate tumor cell proliferation and that this might have a dominant effect over the apoptotic signal.^20-22 In addition, alternative TRAIL signaling in TRAIL resistant cells has been reported.^31,32 In these cells, the predominant effect of TRAIL receptor activation was activation of nuclear factor kappa B (NF-B) leading to cancer promotion rather than inhibition. Further clarification of this complexity of TRAIL signaling, and whether a pro-apoptotic or antiapoptotic pathway is predominant in colorectal cancer, is clearly needed.

High DR5 expression was associated with longer disease-free survival with borderline significance, suggesting an inhibiting effect of DR5 on tumor progression. Recent evidence supports a role for DR5 as candidate tumor suppressor. Silencing of DR5 in human colon cancer cell lines, using inducible RNA interference, led to accelerated growth of bioluminescent tumor xenografts and resistance of tumor cells to fluorouracil.¹⁹

Our findings indicate that DR4 and DR5 expression have an opposite prognostic impact on patient survival. This may be explained by the finding that DR5, and not DR4, is implicated in fluorouracil sensitivity.¹⁹ It has to be kept in mind, however, that only 10% to 15% of stage III colon cancer patients benefit from fluorouracil-based adjuvant chemotherapy.¹ Another explanation may lie in the fact that tumor cells with high DR5 expression seem to be more susceptible to TRAIL-induced apoptosis than tumor cells with high DR4 expression. Under physiologic conditions, TRAIL binds with a higher affinity to DR5 than to DR4.³³ In addition, there is evidence suggesting that DR5 contributes more than DR4 to TRAIL-induced apoptosis in normal and cancer cells that express both receptors.³⁴ Alternatively, DR4 could be nonfunctional in colon cancers because of mutations in or near the TRAIL-binding domain of DR4. DR4 mutations have indeed been found in several cancers, but colorectal cancer has not been studied so far.^35,36

A few studies have investigated the prognostic value of TRAIL, DR4, and DR5 expression in other tumor types. High TRAIL mRNA and protein expression levels were associated with better outcome in ovarian cancer patients.^37,38 DR4 and DR5 expression in 29 acute myelogenous leukemia patients showed no prognostic significance.³⁹ In non–small-cell lung cancer and breast cancer, strong DR5 tumor staining was associated with an adverse prognosis.^40,41

One potential limitation of our study lies in the use of tissue microarrays. Tissue microarrays allow high-throughput analysis of large numbers of samples, but concern has been expressed about the validity of such an analysis, especially in case of tumor heterogeneity. However, the validity and representativeness of this strategy has been demonstrated in several studies (reviewed in Bubendorf et al⁴²). In addition, the prognostic markers studied in this report (DR4 and DR5) are known to show homogeneous expression in colon tumors,¹⁷ which facilitates the use of tissue microarray analysis. TRAIL expression is generally rather heterogeneous in colon tumors. To minimize the influence of this heterogeneity, three samples from each tumor were therefore analyzed. Finally, several tumors from this cohort were previously analyzed for TRAIL, DR4, and DR5 expression using whole tissue sections,⁴³ and the results from the tissue microarray analysis correlated well with these data.

Several clinical phase I-II studies with rhTRAIL and agonistic anti-DR4 and anti-DR5 antibodies are ongoing.^44-47 The success of such therapies will, among other factors, require death receptors on the cell surface. Several anticancer drugs, including fluorouracil, are known to increase DR4 and DR5 membrane expression on the cell surface and to enhance TRAIL-mediated apoptosis in colon cancer cells in vitro.^48-53 In support of further development of rhTRAIL or TRAIL receptor agonistic antibodies as anticancer agents for colon cancer is the finding that most colon tumors showed high expression levels of DR4 and DR5, independent of p53 mutations.

In conclusion, high DR4 expression is associated with worse disease-free and overall survival in adjuvantly treated colon cancer patients. The biologic background for this finding needs further clarification. Evaluation of DR4 expression in stage III colon cancer patients may identify a subset of patients requiring more aggressive adjuvant treatment. The finding of high DR5 expression in the majority of tumors suggests that DR5 agonists may be interesting biologic drugs for colon cancer patients.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Author Contributions

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Conception and design: Caroline M. van Geelen, Jantine L. Westra, Elisabeth G. de Vries, Nanno H. Mulder, John T. Plukker, Steven de Jong, Jan H. Kleibeuker, Jan J. Koornstra Provision of study materials or patients: Jantine L. Westra, Wytske Boersma-van Ek, Nanno H. Mulder, John T. Plukker Collection and assembly of data: Caroline M. van Geelen, Jantine L. Westra, Wytske Boersma-van Ek, Nynke Zwart, Harry Hollema, Steven de Jong, Jan J. Koornstra Data analysis and interpretation: Caroline M. van Geelen, Elisabeth G. de Vries, Harry Hollema, H. Marike Boezen, Steven de Jong, Jan H. Kleibeuker, Jan J. Koornstra Manuscript writing: Caroline M. van Geelen, Elisabeth G. de Vries, Jan J. Koornstra Final approval of manuscript: Caroline M. van Geelen, Jantine L. Westra, Elisabeth G. de Vries, Wytske Boersma-van Ek, Nynke Zwart, Harry Hollema, H. Marike Boezen, Nanno H. Mulder, John T. Plukker, Steven de Jong, Jan H. Kleibeuker, Jan J. Koornstra

 

	NOTES

 
Supported by Grants No. 1998-1660 and 2000-2286 from the Dutch Cancer Society and Grant No. 2001-31 from the Dutch Digestive Diseases Foundation.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted April 3, 2006; accepted August 31, 2006.

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