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© 2003 American Society for Clinical Oncology Prognostic Value of Thymidylate Synthase, Ki-67, and p53 in Patients With Dukes’ B and C Colon Cancer: A National Cancer Institute–National Surgical Adjuvant Breast and Bowel Project Collaborative Study
From the Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; the National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, PA; and the Department of Oncology, Belfast City Hospital, Northern Ireland. Address reprint requests to Carmen J. Allegra, MD, 31 Center Dr, MSC 2440, Bldg 31, Rm 3A-44, Bethesda, MD 20892-2440; email: allegrac{at}navmed.nci.nih.gov.
Purpose: To define the value of thymidylate synthase (TS), Ki-67, and p53 as prognostic markers in patients with stage II and III colon carcinoma. Patients and Methods: We retrospectively analyzed the prognostic value of TS, Ki-67, and p53 in 706 patients with Dukes’ B (291 patients) or Dukes’ C (415 patients) colon carcinoma who were treated with either surgery alone (275 patients) or surgery plus fluorouracil (FU)-leucovorin chemotherapy (431 patients) in National Surgical Adjuvant Breast and Bowel Project (NSABP) protocols C01-C04. All three markers were assayed using immunohistochemical techniques. Results: Using 5 years of follow-up data, our retrospective analysis demonstrated an association between TS intensity (relapse-free survival [RFS]: risk ratio [RR] = 1.46, P = .01; overall survival [OS]: RR = 1.54, P = .002), Ki-67 (RFS: RR = 0.76, P = .05; OS: RR = 0.62, P = .001), and p53 (RFS: RR = 1.49, P = .01; OS: RR = 1.21, P = .18) for RFS and OS. High TS intensity levels and positive p53 staining were associated with a worse outcome. Tumors containing a high percentage of Ki-67-positive cells enjoyed an improved outcome compared with those patients whose tumors contained relatively few positive cells. An interaction with treatment was not identified for any of the markers. Conclusion: This retrospective investigation demonstrated that TS, Ki-67, and p53 staining each had significant prognostic value for patients with Dukes’ B and C colon carcinoma. However, none of the markers could be used to clearly discern groups of individuals who would be predicted to derive greater or lesser benefit from the use of adjuvant chemotherapy.
COLORECTAL CANCER is the third most common cancer in the United States, with an estimated 130,000 new patients diagnosed each year, and is the second leading cause of death because of cancer (approximately 56,000 people annually). About 60% of all patients diagnosed with colorectal carcinoma will present with locally advanced disease. Appropriate therapeutic decision making for these individuals depends primarily on the depth of penetration of the primary tumor and malignant involvement of the regional lymph nodes. Although surgery is the mainstay of therapy for patients with localized disease, standard of practice is to offer adjuvant chemotherapy, particularly to those individuals with lymph-node-positive colon cancer. Whereas 60% to 65% of patients are cured by surgical intervention alone and adjuvant chemotherapy decreases by about 30% the mortality of those who would have relapsed, approximately one third of those treated are ultimately diagnosed with recurrent disease. Thus, under current guidelines, many patients receive adjuvant chemotherapy unnecessarily, while others who might benefit from it are not recommended to receive it. The goal of this investigation was to define a marker, or set of markers, on which therapeutic decisions could be made with greater precision for given individuals. We elected to investigate p53, Ki-67 as a marker of proliferation, and thymidylate synthase (TS) expression because these markers have been demonstrated in a number of studies to have potential value in defining populations of individuals who either may or may not benefit from the use of adjuvant chemotherapy.1 It is also possible that these markers may serve as an adjunct for the prognostication of natural history for those likely to develop advanced colon carcinoma. Of all potential markers that may have prognostic or predictive value for patients with colon cancer, p53 has been the most investigated. Mutations in p53 have been found to occur in 40% to 60% of patients with colon cancer.2 Preclinical investigations have demonstrated that mutant p53 renders malignant cells less sensitive to most chemotherapeutic agents, with the exception of the taxanes, which seem to be indifferent to p53 status.3–5 Given the logistical difficulties and resources associated with direct sequencing of the p53 gene, most investigations have used immunohistochemistry as a means of detecting mutant p53, with the assumption that overexpression of p53 is often associated with a mutation, while the lack of expression is generally indicative of wild-type p53. This assumption has been valid in approximately 60% to 80% of instances in which mutational analysis and p53 detection by immunohistochemistry have been compared.6–9 Whereas numerous investigations have been performed addressing the possible prognostic and/or predictive value of p53 in patients with colon cancer, the majority have been performed in small numbers of individuals and thus suffer from limited statistical power. This issue is further compounded by the availability and use of multiple antibodies, which have varying levels of sensitivity and capacity for detecting mutant protein. In investigations in which at least 100 patients with locally advanced colon cancer have been studied, those in which monoclonal antibodies to p53 (PAB 1801/DO-7/D0–1) were used have generally demonstrated that mutant or overexpression of p53 is associated with a worse clinical outcome.10–18 However, this association has not been a constant finding; several investigations have found either the opposite or no association between the expression of p53 and clinical outcome.19–24 Thus, while the general impression is that the overexpression of p53 is associated with a less-favorable clinical outcome for patients with locally advanced colon cancer, investigations that demonstrate contrary associations indicate that the role of p53 as a prognostic marker requires additional investigation. Flow cytometry has been the technology most commonly used to measure cell cycling activity in patients with colon cancer. Several investigations have found that a high S-phase fraction (> 20%) is associated with a greater probability of recurrence and diminished survival,23,25–27 although this has not been universally observed. Ki-67 is expressed in cells actively engaged in the cell cycle and has also been used as a measure of proliferation in this patient population. Unfortunately, most of these studies are relatively small and have not demonstrated a consistent association with clinical outcome. TS is the primary intracellular target for the fluoropyrimidine class of chemotherapeutic agents that includes fluorouracil (FU) and capecitabine and several new antifolate agents under clinical development.28–31 This enzyme is responsible for the provision of thymidylate required for DNA synthesis and repair. Both preclinical and clinical investigations have demonstrated the importance of intracellular TS levels as a determinant of sensitivity to FU, and multiple clinical investigations have demonstrated an improved response to fluoropyrimidine-containing regimens in patients with low levels of TS in their cancers compared with the response in patients whose cancers overexpress TS.32–36 Investigators throughout the international medical research community have evaluated the prognostic value of intracellular TS levels. Multiple studies have shown that patients with high levels of TS in their cancers have a significantly worse clinical outcome compared with those patients whose cancers have relatively low intracellular levels.37–42 We designed the investigation described here to assess the value of TS, p53, and Ki-67 as potential prognostic markers, either alone or in combination in patients with Dukes’ B and C colon cancer.
The patients enrolled in this trial were drawn from four randomized National Surgical Adjuvant Breast and Bowel Project (NSABP) colon cancer adjuvant treatment trials. The NSABP C-O1 trial evaluated the postoperative addition of immunotherapy (BCG) or systemic chemotherapy (MOF) in the management of resectable colon cancer. Between November 1977 and February 1983, 1,166 patients with Dukes’ B or C colon cancer were randomized. The NSABP C-O2 trial evaluated postoperative additional portal vein infusion of FU and heparin in the management of patients with resectable adenocarcinoma of the colon. Between March 1984 and July 1988, 1,158 patients were randomized. The NSABP C-O3 trial compared adjuvant therapy using leucovorin and FU with MOF chemotherapy in patients with Dukes’ B and C colon cancer. Between August 1987 and April 1989, 1,081 patients were randomized. The NSABP C-O4 trial assessed the efficacy of FU plus leucovorin compared with FU plus levamisole or FU plus leucovorin plus levamisole in patients with Dukes’ B or C colon carcinoma. Between July 1989 and December 1990, 2,151 patients were randomly assigned to the study arms.
Immunohistochemistry Methods
TS IHC Methods
Tissue Evaluation
p53 IHC Method
p53 Tissue Evaluation
Ki-67 IHC Methods
Ki-67 Tissue Evaluation
IHC Negative Controls
Statistical Analysis Marker scores were categorized for analysis purposes in the following manner: positive versus negative for p53; 41% to 100% versus 0% to 40% for Ki-67; diffuse versus focal for TS staining pattern; and bright and/or dark intensity3 versus definite staining of light to moderate intensity or weaker (0 to 2) for TS intensity. The relationship between each marker and outcome was first assessed separately for each treatment group, and further by Dukes’ class. Both treatment groups were combined, and the analyses were repeated. Cox proportional hazards models were used to compare OS and RFS between marker categories. Risk ratios were obtained from these models. Within treatment groups, analyses were stratified by the number of positive nodes (no positive nodes, 1 to 4 positive nodes, 5 or more positive nodes, some positive nodes but exact number unknown, unknown number of positive nodes). In analyses of combined treatment groups, models were stratified by treatment and number of positive nodes. The stratified log-rank test was used to test for differences between marker categories.
Survival distributions were estimated in S-Plus using the Kaplan-Meier method. Global tests for interactions of marker pairs and markers with treatment and the number of positive lymph nodes were conducted using a likelihood ratio test.
Patient Characteristics This study is a cross-protocol retrospective study of 706 of 2,774 patients with Dukes’ B and C colon cancer who were randomly assigned or registered to surgery alone on NSABP protocols C-01 or C-02 or randomly assigned to receive FU and leucovorin following surgery on protocol C-03 or C-04. Two hundred seventy-five Dukes’ B or C surgery-alone patients (32% of those randomly assigned or registered in protocols CO-1 and CO-2) and 431 (23% of those in protocols C-03 and C-04) patients treated with FU/leucovorin had paraffin blocks that were of suitable quality for these analyses. As Table 1  shows, the characteristics of the patients whose tissue we were able to evaluate are quite similar to those of patients not included. In addition, log-rank tests comparing RFS and OS of patients included in the study (706 patients) with those not included in the study (2,068 patients) had P values of .84 and .88, respectively. The 5-year RFS (OS) rates for the included patients versus the excluded patients were 0.69 versus 0.70 (0.69 v 0.71). Thus, there is evidence that the patients included in this study are representative of those participating in the four randomized trials.
Markers Markers were classified into two categories, negative (low) values and positive (high) values (Table 2 ). These categories were chosen to assure that there would be enough patients in each category to permit robust multivariate analyses; any other subgrouping resulted in the smaller set having fewer patients than with the current grouping. The primary analyses were conducted using these categories, although each marker was also analyzed according to the original classifications.
Figures 1A , 1B, 2A, and 2B show RFS and OS, respectively, for Ki-67 and TS intensity using the original classifications, and Figs 3A–D and 4A–D show RFS and OS, respectively, for these two variables plus TS staining pattern and p53 when patients were categorized as shown in Table 2 . Table 3 delineates the relative hazards and associated P values for positive and negative groups for each marker in all patients, and separately for Dukes’ B and C patients. When Ki-67 and TS intensity were analyzed according to the original classification, the P values were similar to those presented in Table 3.
In terms of the relation between individual markers and RFS and OS, we found no statistically significant interactive effects between treatment and marker values, indicating that there is no evidence of a differential treatment effect according to whether a marker is positive or negative (RFS, P = .92; and OS, P = .59). A global test for the interaction of the markers with Dukes’ class was significant both with respect to RFS (P = .001) and to OS (P = .001). These observed differential prognostic effects by Dukes’ class may be caused by chance but are presented in detail below for others to refute or confirm. The global test for between marker interactions with outcome was not significant for RFS (P = .78) or OS (P = .98), indicating that one marker does not influence the prognostic effect of another marker.
TS Staining Intensity Because some previous studies have reported a possible TS–treatment interaction, we performed several secondary analyses to investigate this question in our patient population.42,47 We did a formal test of interaction of TS level with treatment (with FU/leucovorin) in which we defined the high-TS-intensity group by patients categorized as a 3. We then fit a Cox model with treatment, TS, and their interaction term in a model. The interaction term had a P value of .24 for RFS and .67 for survival. We also considered using a lower cutoff and grouping TS intensity by 0 to 1 versus 2 to 3. Using this method of categorization, the tests for interaction with treatment effect had P values of .17 for RFS and .41 for survival. We were unable to identify any subgroup that failed to benefit from FU/leucovorin therapy.
TS Staining Pattern
Ki-67
p53
Association of Markers
Multivariant Analysis When all markers were included as independent variables in a Cox proportional hazards model (stratified by treatment and lymph nodal status), the presence of p53 (P = .01) and increased TS intensity (P = .04) were both determined to be significantly associated with poor RFS. Larger values of Ki-67 were associated with a marginally better RFS (P = .03), and smaller values of Ki-67 (P = .001) and increased TS intensity (P = .02) both were determined to be significantly associated with poor OS.
Combining Markers
In this investigation we included tumor samples from a total of 706 patients who had been entered onto four randomized NSABP protocols, including 275 colon cancer patients with Dukes’ B and C treated with surgery alone, and 431 treated with surgery followed by weekly bolus FU plus leucovorin therapy. Evidence that the subset of patients used for the marker studies was representative of the total treatment population is indicated by the nearly identical 5-year RFS and OS demonstrated by these two groups as well as by patient characteristics including stage, sex, and age, which were indistinguishable between the two groups. The central finding of this investigation is that high TS intensity, p53 positivity, and low levels of Ki-67 nuclear staining were each significantly associated with a poor 5-year RFS and/or OS. These associations were similar for Ki-67 and TS intensity whether these variables were treated as dichotomous variables or were analyzed according to their original classifications. A high level of TS staining intensity was associated with a relative risk of approximately 1.5 for both RFS and OS. Positive p53 associated with a relative risk of approximately 1.5 for RFS and 1.2 (not statistically significant) for OS. A high level of Ki-67 nuclear staining, that is, greater than 40% of cells positive, was associated with relative risk of 0.76 and 0.62 for 5-year RFS and OS, respectively. In the multivariate analysis that included treatment and Dukes’ stage, high TS scores and smaller values of Ki-67 were both associated with a significantly poorer OS, while high TS scores and p53 positivity were each associated with a significantly poorer RFS. Smaller values of Ki-67 were associated with a poorer RFS. We also found that TS intensity was negatively associated with Ki-67 nuclear staining. Given that the intracellular level of TS is related to cell cycling, it was somewhat surprising to find this negative association with Ki-67, a marker of proliferation. This negative association suggests that the prognostic significance of TS transcends that of being a simple marker of proliferation. Our data that demonstrate an interaction between TS staining intensity and clinical outcome are in concert with the preponderance of published literature that addresses the prognostic significance of TS intensity in patients with Dukes’ B and C colorectal carcinoma.37–42 TS was first demonstrated to have prognostic value in patients with locally advanced rectal cancer treated on NSABP Protocol R-01, which was designed to test the utility of the addition of MOF chemotherapy or radiation therapy to standard surgical resection in 300 patients with rectal cancer.37 This study used the monoclonal antibody TS 106 and demonstrated that patients whose tumors contained high levels of TS had a worse clinical outcome compared with those whose tumors contained low levels of TS. A later investigation by Lenz et al38 measured TS levels in 45 patients with stage II colon cancer and also found that those patients who overexpressed TS had a significantly worse outcome compared with those who had relatively low levels of TS in their cancers. Multiple subsequent investigations have confirmed the independent prognostic value of TS in patients with locally advanced colon cancer.39–45 One notable exception is the North Central Cancer Treatment Group (NCCTG) study of 465 patients with stage II and III colon cancer in which TS intensity was not found to be a significant prognostic indicator.46 In this study, almost 70% of patients were treated with various systemic FU-based chemotherapeutic regimens that included leucovorin and/or levamisole or interferon-gamma or intraportal vein FU. Although using a TS intensity assessment identical to this study did demonstrate that patients included in the NCCTG study with high levels of TS had a worse prognosis than those with low intensity, this difference did not reach statistical significance. Why the NCCTG study did not demonstrate the prognostic value of TS is not clear; however, one possible explanation may be the use of various chemotherapeutic regimens, most of which included levamisole, administered by the systemic or portal route and the unconfirmed observation made by Edler et al42 that patients with low-intensity TS levels actually had a worse outcome when treated with chemotherapy (50% treated with levamisole-containing regimens) compared with patients who were treated with surgery alone. If true, such a treatment interaction would tend to decrease any differences in outcome between patients with high versus low TS. Given that almost 70% of the patients included in the NCCTG study received some form of chemotherapy, it is possible that the prognostic value of TS could not be discerned if one or several of the chemotherapeutic regimens, perhaps those that included levamisole, in fact improved outcome specifically in patients with high TS intensity. No such interaction between TS intensity and chemotherapy was identified in this study; however, our investigation excluded those patients who received levamisole-containing regimens. Although the prognostic value of p53 overexpression demonstrated by immunohistochemistry is controversial for patients with locally advanced colorectal cancer, our data support the preponderance of clinical trials that use a monoclonal antibody for assessing p53 and contain at least 100 patients. In general, such studies have demonstrated that overexpression of p53 is associated with a worse clinical outcome.10–18 Clearly, this association has not been found to be universally true, as several investigations, including a recent study by Watanabe et al,47 failed to demonstrate a significant association between p53 overexpression and clinical outcome in patients with locally advanced colon cancer. At least two investigations have shown an improved clinical outcome for patients whose tumors overexpress p53.23,24 These two investigations used the monoclonal antibody DO7 and in each instance found a statistically significant improvement in survival for those patients with stage III disease whose tumors demonstrated overexpression of p53. Neither study demonstrated improvement in outcome with p53 overexpression in patients with Dukes’ B colon cancer. Our finding that high levels of nuclear staining with Ki-67 are associated with improved outcome seems counterintuitive, given that several, but not all, investigations that used flow cytometry to measure proliferative rate in patients with colon cancer found that a high S-phase fraction was associated with a worse clinical outcome.23,25–27 This inverse association was true even when Ki-67 was analyzed according to its original grouping. The biologic reason as to why high levels of Ki-67 should be associated with improved outcome will require additional preclinical and clinical investigations. When we performed a global test for interaction of the various markers with Dukes’ class, we found a significant interaction, both for RFS and OS. The significance of this global interaction was primarily because of Ki-67 in patients with Dukes’ B disease. Dukes’ B patients with high levels of Ki-67 demonstrated a poorer outcome with respect to RFS and OS; the reverse was true in patients with Dukes’ C disease. The reversal of prognostic significance for Ki-67 with respect to stage is perplexing, and the explanation for this reversal is not obvious. It is possible that this observation may be a result of chance. Thus, further investigations will be required to clarify this particular observation. One of the goals of our study was to try to identify, based on the three markers studied, subsets of individuals who may demonstrate greater or lesser benefit from the use of adjuvant chemotherapy. However, our analysis demonstrated no significant interaction between treatment and the various marker values. Thus, patients in each of the marker categories derived equal benefit from the use of adjuvant chemotherapy. Whereas wild-type p53 (ie, negative p53 staining and high proliferative rates) would be expected to be associated with chemotherapeutic responsiveness, we were able to identify no such associations. Possible explanations include the use of p53 immunohistochemistry that reflects the mutational status of p53 in only 60% to 80% of patients6–9 and the possibility that relatively small differences could have been missed because of the power of the study. Furthermore, although Ki-67 is a reflection of the percentage of cells actively cycling, it does not necessarily quantitate the rapidity of the cycling process that may be the critical factor in cell sensitivity.48 Thus, it is conceivable that even cancers with relatively high levels of Ki-67 staining may have relatively slow doubling times. It was somewhat surprising that TS levels did not predict benefit from the use of adjuvant chemotherapy, given the consistent finding in patients with advanced colorectal cancer in whom high TS levels were predictive for nonresponsiveness to fluoropyrimidine-based therapy.32–36 One possible explanation for this apparent discrepancy may be that the TS levels in the primary cancer are not reflective of the levels in micrometastases that would represent the mechanism of relapse. Reports have demonstrated that TS levels in the primary cancer are not related to the levels in matched samples taken from metastatic deposits.49 Recent investigations in patients with locally advanced colorectal cancer have found conflicting results with respect to a treatment interaction in patients whose cancers have low levels of TS. One group of investigators found that patients whose tumors have low TS levels derive greater benefit from adjuvant chemotherapy compared with those whose tumors have high TS levels.50 A second group found the exact opposite to be true; that is, patients whose cancers had low TS levels suffer an adverse effect from the use of adjuvant chemotherapy.42 A potential reason for this marked discrepancy may be the use of different technologies for the assessment of intratumoral TS levels. Thus, although our study did not identify TS as a predictive marker, whether this enzyme has potential value as a predictor of benefit from adjuvant chemotherapy in patients with locally advanced colorectal cancer remains unclear. In a further attempt to identify subsets of patients who had a particularly good or poor clinical outcome, we combined two or three markers. Overall, we found that patients whose tumors had a high Ki-67, low TS intensity, and were negative for p53 had a 5-year RFS of 0.80, and those with the opposite characteristics had a 5-year RFS of 0.51, indicating that these markers may be used to identify individuals who are at particularly high risk for relapse and death from their colon cancer. Given the uncertain prognostic value of Ki-67, we also performed analyses using only TS intensity and p53 positivity and found that patients whose tumors had a high level of TS intensity and were positive for p53 had a 5-year RFS of 0.51 versus those with the opposite characteristics who had a 5-year RFS of 0.75 if they had lymph-node-positive disease. Because the 5-year rates were determined using the same data that were used to identify the two extreme subgroups, it is likely that the differences between the rates are an overestimate, so these results need to be confirmed in subsequent studies. However, they do suggest that Ki-67, TS intensity, and p53 may play a role in identifying individuals who are at a particularly high risk for relapse and death from their colon cancer. In summary, we demonstrated that each of the three markers investigated, TS, p53, and Ki-67, carries prognostic significance with respect to RFS and OS for patients with Dukes’ B and C colon carcinoma. However, none of the markers could be used to clearly discern groups of individuals who would be predicted to derive greater or lesser benefit from the use of adjuvant chemotherapy.
We thank Carol Ursiny, CCRA, for her contribution in maintaining the NSABP colon cancer trials database, and Barbara C. Good, PhD, for editorial assistance.
Supported by Public Health Service grants U10CA12027, U10CA69651, UC10CA37377, and U10CA69974 from the National Cancer Institute (NCI), National Institutes of Health, Department of Health and Human Services, Bethesda, MD. This investigation was conducted after approval by a local and NCI Institutional Review Board committee and in accord with an assurance filed with and approved by the Department of Health and Human Services. An abstract of this information was presented at the 2001 meeting of the American Society of Clinical Oncology as "Prognostic Value of Thymidylate Synthase, Ki-67, and p53 in Patients with Dukes’ B and C Colon Cancer."
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 tests were used to assess the relationship between marker pairs. 
