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Investigation of the Prognostic and Predictive Value of Thymidylate Synthase, p53, and Ki-67 in Patients With Locally Advanced Colon Cancer

From the National Cancer Institute, Bethesda, MD; Mayo Clinic and Mayo Foundation, Rochester, MN; Meritcare Hospital Community Clinical Oncology Program, Fargo, ND; Illinois Oncology Research Association Community Clinical Oncology Program, Peoria, IL; Saskatoon Cancer Centre, Saskatoon; and Allan Blair Cancer Centre, Regina, Saskatchewan, Canada.

Address reprint requests to Richard M. Goldberg, MD, Mayo Clinic, 200 1st St. SW, Rochester, MN 55905; email: goldberg.richard@ mayo.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate the value of thymidylate synthase (TS), Ki-67, and p53 as prognostic markers in patients with Dukes’ B2 and C colon carcinoma.

METHODS: We conducted a retrospective analysis to evaluate the prognostic value of TS, Ki-67, and p53 in 465 patients with Dukes’ B2 (220 patients) or Dukes’ C (245 patients) colon carcinoma. Patients represent a nonrandom subset obtained from five randomized phase III trials and were treated with either surgery alone (151 patients) or surgery plus fluorouracil-based chemotherapy (314 patients). All three markers were assayed using immunohistochemical techniques.

RESULTS: With a minimum follow-up of 5 years, our retrospective analysis failed to demonstrate a consistent and significant association between TS, Ki-67, or p53 and either disease-free survival or overall survival. Exploratory analyses did not reveal a convincing explanation for these results that are in conflict with the published literature. Notable interactions were observed. In particular, high Ki-67 levels were associated with increased (decreased) survival in patients with low (high) TS intensity. Patients whose tumors stained positively for p53 seemed to benefit substantially from the use of adjuvant chemotherapy compared with those who were not treated (P = .05).

CONCLUSION: This retrospective investigation failed to demonstrate a significant association between TS, Ki-67, or p53 staining and clinical outcome.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
COLORECTAL CARCINOMA is the second most common form of cancer in the United States and accounts for the death of over 55,000 individuals annually (Surveillance, Epidemiology, and End-Results database). At the present time, the therapeutic decision to use adjuvant chemotherapy after curative intent surgery depends primarily on the depth of tumor penetration into the bowel wall and the status of the lymph nodes with respect to carcinomatous involvement. Given that approximately 60% to 65% of patients with Dukes’ B2 and C colon cancer are cured with surgery alone and many patients with Dukes’ B2 disease are not treated with adjuvant chemotherapy, many patients receive chemotherapy unnecessarily whereas others who are not treated may benefit from its use. The goal of the present investigation is to explore possible prognostic and predictive markers so that therapeutic decisions may be made with greater precision, thus focusing the use of adjuvant chemotherapy primarily in those individuals most likely to derive benefit. Many putative prognostic markers have been investigated in patients with colorectal cancer over the past several decades.¹ Perhaps the most frequently studied have been p53 mutational status and measures of cell cycling. More recently, thymidylate synthase (TS) has been investigated as both a prognostic marker and as a predictor of response to fluoropyrimidine-based therapies that target TS as their primary mechanism of action.

p53 has been referred to as the guardian of the genome for its importance and central role in controlling both the cell cycle and the apoptotic machinery.² Mutations in p53 occur in approximately half of all cancers, and in general, cancer cells with mutations in p53 are less sensitive to most commonly used chemotherapeutic agents.^3-5 One major exception is the taxanes, where sensitivity to this class of agents is relatively unaffected by p53 mutational status.³

Most investigations of p53 as a prognostic indicator have used immunohistochemical (IHC) techniques with the assumption that overexpression is a surrogate for a mutation. This assumption is imperfect in that IHC is concordant with the mutational analysis in only 65% to 80% of cases.^6-9 In general, reactivity to the polyclonal antibody CM1 has not been found to be a useful prognostic indicator of clinical outcome. However, in studies that use monoclonal antibodies to p53 (PAB 1801/DO-7/D0-1) and included at least 100 patients with Dukes’ B and/or C colon cancer, overexpression of p53 was associated with a worse disease-free survival (DFS) and/or overall survival (OS) in nine of 15 published series.^10-18 Five studies demonstrate no association^19-23 between p53 expression status and clinical outcome, and two demonstrate an improved clinical outcome for patients with p53 overexpression.^24,25 Of particular interest is a report in which Soong et al²⁵ used both IHC with the monoclonal antibody D07 and direct sequencing of p53 in 541 patients with colorectal cancer. They noted an improved survival for those patients who overexpress p53, specifically in patients with distal colonic primaries and Dukes’ C disease, but not for tumors located in the proximal colon or Dukes’ B-stage disease.²⁵ Mutations in p53 detected by direct sequencing showed similar trends as noted with IHC.

Thus, although the weight of the published literature suggests that overexpression of p53 is associated with poorer outcomes in patients with local or locally advanced colorectal cancer, contradictory reports suggest that the clinical relevance of p53 overexpression in this patient population remains unclear. The lack of clarity may be attributable to the limited detection power inherent in studies that test small subsets of patients and the diversity inherent with the inclusion of patients with all Dukes’ stages in the analysis.

The MIB-1 antibody that recognizes an epitope on Ki-67 has been frequently used as a measure of proliferation because this antigen is expressed only in cells actively engaged in the cell cycle. Flow cytometry has also been used to measure the proportion of cells in S phase, and this technique has most commonly been applied to determine the prognostic value of cellular proliferation in patients with colorectal carcinoma. Several studies measuring proliferative activity in the cancers of patients with Dukes’ B2 and C colorectal carcinoma used flow cytometry and found that a higher proliferative index ( 20) predicted for a higher probability of recurrence and diminished survival.^26-28 However, measures of proliferation have not always demonstrated a significant association with clinical outcome.^24,29,30

TS is an enzyme responsible for the catalytic methylation of deoxyuridylate to thymidylate, which is required for DNA synthesis and repair. Given its central role in pyrimidine metabolism, this enzyme has been the target of cancer therapeutics for over four decades. The fluoropyrimidine class of anticancer agents target this enzyme as their primary mechanism of action, and a new class of antifolate compounds that specifically target TS are under investigation as therapeutic agents.^31-34 Several preclinical and clinical investigations have demonstrated that the relative levels of TS are a critical determinant of cellular sensitivity to agents that target TS. Given the central role of TS in the metabolic pathways and as a therapeutic target, several investigations have addressed the potential importance of the relative levels of this enzyme as both a prognostic factor and as a predictor of response to agents that target TS. All studies to date show that high levels of TS correlate with lower response rates in patients with advanced disease treated with fluoropyrimidine-based therapy. High TS has also been shown to be associated with a worse clinical outcome for patients with locally advanced colon and rectal cancer. Because biochemical assays that measure TS are relatively insensitive, highly sensitive IHC and reverse transcriptase polymerase chain reaction techniques have been developed for use in measuring the level of TS in patient tumor samples.^35,36 These two methods of assessing TS levels have been compared and found to closely parallel one another with respect to the levels of TS and as predictors of clinical outcome in patients with gastrointestinal cancers treated with fluorouracil (5-FU)-based therapies.³⁷

The present investigation was designed to assess the value of TS, p53, and Ki-67 as prognostic markers when used alone or in combination in patients with Dukes’ B2 and C colon cancer in a group of patients treated as part of the several clinical trials conducted by the North Central Cancer Treatment Group (NCCTG).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The patients enrolled onto this trial were drawn from the following five NCCTG colorectal cancer treatment trials: (1) NCCTG 78-48-52, an evaluation of immunotherapy or chemo-immunotherapy as adjuvant treatment in resectable adenocarcinoma of the colon or rectum, which enrolled 401 patients who were randomly assigned to 5-FU plus levamisole, levamisole alone, or no adjuvant therapy after resection of Dukes’ B2 or C colon cancer³⁸; (2) NCCTG 79-46-04, adjuvant therapy of resectable adenocarcinoma of the colon with 5-FU administered by portal vein infusion that enrolled 224 patients randomly assigned to postoperative intraportal 5-FU for 7 days or to no treatment³⁹; (3) NCCTG 84-46-52 (Intergroup 0035), an evaluation of levamisole alone or levamisole plus 5-FU as surgical adjuvant treatment for resectable adenocarcinoma of the colon, which entered 1,296 patients who were randomly assigned to 5-FU plus levamisole, levamisole alone, or no adjuvant therapy after resection of Dukes’ B2 or C colon cancer⁴⁰; (4) NCCTG 87-46-51, a controlled evaluation of recombinant interferon-gamma and 5-FU and folinic acid (ie, leucovorin) as adjuvant treatment for resectable adenocarcinoma of the colon, which entered 160 patients to receive either interferon-gamma injections, 5-FU plus leucovorin, or no treatment after resection of colon cancer⁴¹; and (5) NCCTG 89-46-51, a controlled phase III evaluation of 5-FU combined with levamisole and leucovorin as adjuvant treatment for resectable colon cancer, which randomized 915 patients to 5-FU and levamisole plus or minus leucovorin for either 6 or 12 months.⁴²

Patients were enrolled onto these five trials from November 1979 through October 1991. A total of 1,524 patients of 1,892 eligible patients randomized to the above noted trials were considered eligible for this study; patients randomized to the levamisole alone arms or Dukes’ B2 patients without risk markers⁴³ were excluded from this analysis. Paraffin-embedded tumor tissue was submitted for 545 of these patients (36%), of whom 15% (80 of 545) had insufficient tumor tissue for analyses (ie, the block contained less than 25% tumor) of immunohistochemical parameters (IHPs), resulting in an analysis data set of 465 patients. These patients represent a nonrandom subset of those enrolled onto these trials.

Patient Follow-Up
Patients were observed for a minimum of 8 years for OS, with a minimum follow-up of 12 years on trials 78-48-52 and 79-46-04. OS is calculated as the number of days from randomization to death or date of last contact in those patients lost to follow-up. Patients were observed routinely for 5 to 8 years after randomization for disease recurrence on each of the five treatment trials. The disease-free interval [ie, DFS] is calculated as the number of days from randomization to the date of recurrence or death. OS for patients on the two older studies (78-48-52 and 79-46-04) were censored at 12 years, with censoring at 8 years on the three more recent studies. All patients were censored at 5 years after randomization for DFS.

IHC Methods
Blocks were processed at the NCCTG tissue repository, and five individual 6-micrometer sections were mounted on glass slides. Each slide was identified by a unique number blinded with respect to patient identity and clinical characteristics.

TS IHC Methods
Tissue specimens were first deparaffinized in 100% xylene and rehydrated through graded alcohol solutions. They were next rinsed in phosphate-buffered saline (PBS) for 5 minutes, then in dH₂O for 5 minutes. Endogenous peroxidase activity was inhibited by incubating the slides in 3% H₂0₂ for 10 minutes, followed by a 5-minute rinse in dH₂O, and finally a 5-minute rinse in PBS. The slides were then loaded onto the Optimax Plus automated cell stainer (BioGenex, San Ramon, CA). The tissues were blocked with horse serum for 30 minutes to reduce nonspecific staining, then incubated for 50 minutes with TS primary antibody at a 1:500 dilution. The slides were washed four times with Optimax wash buffer (BioGenex) and then incubated in biotinylated goat antimouse secondary antibody for 30 minutes. The slides were again washed four times with Optimax wash buffer and then incubated with avidin-biotin complex (ABC) for 30 minutes. After washing, the chromagen, diaminobenzidine (0.7 mg/mL), was applied for 4 minutes. After another wash cycle, the tissues were counter-stained with Mayer’s hematoxylin. The slides were rinsed, dehydrated in graded alcohol solutions, fixed in xylene, and finally mounted on glass coverslips with Permount (ProSciTech, Kelso, Queensland, Australia). To ensure consistent staining, a slide of an adenocarcinoma sample was included in each staining run and scored.

TS Tissue Evaluation
Each slide was assigned a score for intensity and staining pattern. Intensity scores range from 0 to 3, and the staining pattern was either F (focal) or D (diffuse). We used two separate scoring systems. In the first method (method A), the intensity scale was 0 = no staining, 1 = weak to light moderate staining, 2 = moderate intensity, and 3 = bright and/or dark intensity. If 20% of the malignant cells were stained at the assigned intensity level, the staining pattern was scored as F. If > 20% were stained, the slide was scored as D. In the second method (method B), the slides were again assigned intensity and staining pattern scores. For intensity, 0 = no staining, 1 = trace staining, 2 = definite staining of light to moderate intensity, and 3 = bright and/or dark intensity. In the second method, slides with 50% or fewer of malignant cells stained at the assigned intensity level were considered F, whereas those with greater than 50% stained were scored as D. All specimens were analyzed by two separate investigators (A.L.P. and C.J.A.) who were blinded to all clinical information. Discrepant scores (approximately 25% and 15% of cases with methods A and B, respectively) were resolved by consensus.

p53 IHC Methods
Tissues were deparaffinized, and peroxidase activity was blocked, as described above. After the PBS rinse, antigen retrieval was accomplished by microwaving the slides in a 10-mmol/L citric acid buffer for 3 x 3 minutes. The slides were cooled for 20 minutes on ice before being rinsed in PBS and loaded on the Optimax cell stainer. After the blocking step, the slides were incubated with the p53-D07 primary antibody (Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom) at a 1:50 dilution for 1 hour. After the secondary antibody and ABC solution incubations, the slides were incubated with diamino benzoate for 12 minutes. The slides were mounted as described above. As a positive control, an adenocarcinoma sample known to positively stain for p53 was included in each staining run.

p53 Tissue Evaluation
Using a light microscope, a visual grading system based on the number of positively stained nuclei of the malignant cells in each tissue was used. If 10% or more of the malignant nuclei were stained, the slide was scored as positive. If fewer than 10% of the nuclei were stained, the slide was scored as negative. All specimens were analyzed by two separate investigators (A.L.P. and C.J.A.) who were blinded to all clinical information. Conflicts in scores (approximately 5% of cases) were resolved by consensus.

Ki-67 IHC Methods
Tissues were deparaffinized, and peroxidase activity was blocked, as described above. Antigen retrieval was accomplished by microwaving the slides in citric acid buffer for 4 x 3 minutes plus 1 minute cooling. After cooling on ice, the slides were loaded on the Optimax cell stainer. After the blocking step, the slides were incubated with the Ki-67 MIB-1 primary antibody (Gallus Immunotech, Wildwood, MO) at a 1:50 dilution for 1 hour. After incubating with secondary antibody and ABC, the slides were incubated with diamino benzoate for 12 minutes. The slides were mounted as described above. An identically processed slide containing tissue from a high-grade lymphoma was included in each staining run as a positive control.³⁹ To control against the possibility of nonspecific staining, each case was stained with a nonspecific mouse immunoglobulin G as a primary antibody (Vector Laboratories, Burlingame, CA) at a 1:10,000 dilution. Each slide was analyzed to verify that positive staining was absent.

Ki-67 Tissue Evaluation
Each slide was scored based on the percentage of positively stained malignant nuclei. The following ranges were used: 0% to 20%, 21% to 40%, 41% to 60%, 61% to 80%, and 81% to 100%. The Ki-67 antibody staining intensity and patterns were analyzed by two observers (A.L.P. and C.J.A.) who were unaware of patient identity. Any scoring discrepancies (approximately 15% of cases) were resolved by consensus.

Statistical Methods
The sample size for this study was prospectively determined to provide 90% power to detect a two-fold difference in the hazard rate between low and high TS patients that were treated with either a 5-FU–based regimen or untreated postoperatively. The study design required 275 blocks from patients treated with 5-FU–based therapy and 175 blocks from patients having no adjuvant treatment, which based on a 65% 5-year survival in patients with low TS levels, would result in 122 and 115 events to provide 90% power, respectively. The distributions of OS and 5-year DFS were estimated using Kaplan-Meier⁴⁴ methodology; univariate associations were tested using the log-rank statistic. Cox proportional hazards modeling⁴⁵ was used to explore the association of clinical characteristics and IHP reads, as well as the possible interaction of these factors with OS and DFS. Models were stratified according to the treatment study to which the patient was initially randomized. The score statistic was used to test for the significance of a covariate in a univariate Cox model. The likelihood ratio test was used to test the significance of covariates in the multivariate setting. Graphical methods were used to examine whether underlying model assumptions were satisfied (eg, proportional hazards).⁴⁶ Analyses investigating IHP by treatment interactions included only those patients (n = 314) entered onto randomized studies of treatment versus no treatment. Analyses were performed using SAS (SAS Institute, Cary, NC).⁴⁷ All P values reported are two-sided, and values less than .05 are considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Patient demographics for 465 patients (n = 314 for adjuvant 5-FU–based treatment and n = 151 for no adjuvant treatment) in this analysis are listed in Table 1. Eighty-four percent of patients in both groups were classified as Dukes’ C. Sex, number of nodes, location of tumor, patient age, and grade were not significantly different between patients used for this study versus those entered onto the treatment trials from which these patients were drawn.

Methods A and B for TS scoring were then correlated with each other. In 133 cases (55%), tissue that method A scored as 0 or 1 (ie, low TS) were upgraded to 2 or 3 (ie, high TS) for intensity; whereas only six cases (3%) were down-graded to low (ie, 0 or 1) with respect to TS intensity. 51% of cases (235 of 465) remained the same using either method with respect to TS staining pattern.

Correlation of IHP With Patient Characteristics
Patient characteristics at study entry were correlated with each IHP. Older patients (age over 70 years) tended to have high Ki-67 levels (P = .02). p53 positivity (overexpression) was observed more frequently in distal tumors (P = .02). No other significant correlations between IHP and patient characteristics were observed.

Relationship Between IHP, Survival, and DFS
Two hundred twelve patients (46%) have died with a minimum follow-up of 5 years. Thirty-six percent of patients (163 of 465) had documented recurrent disease within 5 years of initial randomization. Table 3 lists the estimated median OS and univariate risk ratios for patients classified by IHP categories. Based on scoring system A, a diffuse TS staining pattern was associated with a lower hazard of death relative to patients having focal TS staining pattern (HR = 0.7; 95% confidence interval [CI] 0.5 to 1.0; P = .04). This association was not maintained using method B scoring system. No other IHP was significantly associated with OS or DFS.

In exploratory analyses, we investigated models using multiple IHPs and models with interactions between the different IHPs. All of the analyses were adjusted for Dukes’ stage and the use of adjuvant chemotherapy. Several notable interactions were observed. Increased survival (P = .002) and DFS (P = .003) was observed in patients having high TS intensity and diffuse TS staining (based on method A TS reads only) compared with those without both characteristics. This interaction was not maintained using method B TS reads.

An interaction was also observed between TS intensity based on the method A scoring algorithm and Ki-67. High Ki-67 levels were associated with increased survival in patients with low TS intensity, however high Ki-67 levels were associated with poorer survival in patients with high TS intensity (P = .03 for OS and P = .003 for DFS). This association was not maintained at a high level of significance under the method B TS scoring system (P = .25 for OS, P = .07 for DFS) (Fig 1).

View larger version (17K): [in this window] [in a new window]   Fig 1. Each panel represents the 5-year OS of patients as assessed using the various IHC parameters, including TS intensity pattern, and using both methods A and B, p53, and Ki-67.

Relationship Between IHP and Treatment Efficacy
Multivariate models were used to explore the relationship between the benefit of adjuvant treatment and the various IHP. In this analysis, we focused on the Dukes’ C patients, because adjuvant 5-FU–based therapy is the current standard of care for those patients, and only included the 245 Dukes’ C patients who were randomized to a treatment (n = 113) versus no treatment (n = 132) comparison. No significant interaction between benefit of treatment and TS intensity using either scoring algorithm was observed. However, those patients with a focal pattern of TS staining as assessed using method B seemed to have an improved DFS (HR = 0.5, P = .02) with treatment compared with those patients who did not receive chemotherapy. Their 5-year survival was marginally improved (HR = 0.6, P = .08). A treatment by p53 interaction was also observed. 5-FU–based adjuvant treatment was associated with a higher risk of death (ie, harmful) in patients with normal p53 (HR = 2; 95% CI, 1.2 to 3.6; P = .01), whereas patients classified as p53 positive (overexpressed) benefited significantly from 5-FU adjuvant treatment, relative to those not treated (HR = 0.6; 95% CI, 0.4 to 1; P = .05) (Fig 2). A significant benefit or adverse impact of adjuvant treatment was not observed is any other IHP-based subgroup.

View larger version (26K): [in this window] [in a new window]   Fig 2. Panels A and B represent the overall 5-year survival of patients with Dukes’ C stage colon cancer with either p53 overexpression (A) or no p53 expression as demonstrated by immunohistochemistry (B) as a function of treatment with either surgery alone or surgery plus adjuvant chemotherapy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Using method B to assess TS intensity, we found that those patients with high TS levels did have a worse outcome than those with low levels; however, this difference did not reach statistical significance. In an effort to further explore the reason(s) for the lack of association between TS and outcome noted in the present investigation versus prior publications, we separately analyzed the prognostic value of TS intensity for each of the five clinical trials included in our report. Using either method A or B, we noted that the relationship between TS intensity and clinical outcome in the two oldest studies (78-48-52 and 79-46-04) seemed to be different from the more recent investigations. In these two older studies, patients with low levels of TS staining seemed to do worse than those with high levels of staining. In each of the three more recent clinical trials, patients with low TS levels consistently had better clinical outcomes than those with high TS levels; however, these differences did not reach statistical significance. The reason for this finding is not apparent; however, the overall analysis included a substantial minority of patients from the early trials (37%), suggesting the possibility that a factor such as the age, storage, or processing of the older specimens may have played a role in the apparent lack of association between TS intensity and clinical outcome in this study. Based on the greater ease of distinguishing amongst the TS staining intensity categories using method B, we felt that method B was an improvement over method A.

With regard to TS pattern, TS diffuse staining pattern was associated with a significantly improved OS and DFS compared with those patients having a focal staining pattern in a univariate analysis; however, this was only true using the method A scoring system. This pattern was similar using the method B scoring system; however, the outcome differences were not statistically significant.

As is consistent with previous reports, we found no association between Ki-67 scores and clinical outcome. However, high Ki-67 scores were associated with low TS intensity using either method A or B. Given that the intracellular levels of TS are related to proliferative rate, it is interesting to note that low, rather than high TS intensity, was associated with high Ki-67 staining, thus suggesting that TS is not a reflection of the fraction of cells engaged in the cell cycle as is the case with Ki-67. In concert with several prior studies, p53 positivity was not found to be associated with clinical outcome;^19-22 however, p53 positivity (overexpression) was found to be more common in distal tumors. Lenz et al⁴³ found that overexpression of both TS and p53 occurred more commonly in patients with distal tumors and postulated that this may be one reason for the relatively poorer survival of these patients. In addition, these investigators also used IHC to assess the expression of p53 and found an association between overexpression of p53 and the high levels of TS, an association not found in the present investigation.

Given that intratumoral TS levels, p53 status, and proliferative rate may all impact sensitivity to fluoropyrimidine-based chemotherapy, we investigated the interaction of each of these factors with 5-FU–based adjuvant treatment of 5-FU plus either levamisole or leucovorin, or in one instance, 5-FU administered via a portal vein infusion. We found no statistically significant interaction between treatment and either TS intensity or Ki-67. However, patients whose tumors demonstrated p53 positivity benefited significantly from adjuvant chemotherapy when compared with those who were not treated with adjuvant chemotherapy. Patients whose tumors did not express p53 had a two-fold worse outcome with chemotherapy compared with those patients who were not treated. These data are in contrast with those reported by Ahnen et al,²⁴ who found that p53 overexpression as assessed by IHC (DO-7) in 163 patients with Dukes’ C colon cancer was associated with an improved 5-year survival (56% v 43%, P = .01), and patients whose tumors were negative for p53 seemed to enjoy a greater benefit from adjuvant therapy with 5-FU and levamisole than patients who were positive for p53 (64% v 26%, respectively). These authors did not find p53 to have prognostic value in patients with Dukes’ B disease, regardless of whether they received adjuvant chemotherapy.²⁴ Although our result seems counter intuitive, it is conceivable that p53 positivity, which would generally be considered to have a negative impact on chemotherapeutic sensitivity, may be acting as a marker of cellular processes yet to be defined that may be sensitizing these tumors to the effects of 5-FU–based chemotherapy. We found that positive p53 staining was associated with high levels of Ki-67, which in turn were associated with low TS staining intensity. Both of these factors, high proliferative rate and low TS levels, would be expected to be associated with cellular sensitivity to fluoropyrimidines. Furthermore, p53 positivity in our study was associated with stage C disease, a stage that has been reproducibly demonstrated to benefit from chemotherapy.

In an effort to define subpopulations of patients with a more or less favorable outcome using more than a single marker, we investigated the value of combining two or three markers. In models using multiple markers, we found no subgroups of individuals that were clearly defined as having a consistently worse or better clinical outcome. However, high Ki-67 levels were associated with an improved OS in those patients with low TS intensity (method A only), whereas it was associated with a worse outcome in those patients with high TS (method A only). These data are in concert with a previous publication demonstrating an improved outcome in patients with high Ki-67 and low TS intensity scores.⁵⁵

In summary, this report addresses the potential value of TS, Ki-67, and p53 as prognostic markers in 465 patients with Dukes’ B and C colon cancer. In this investigation we did not identify a clear association between either TS staining intensity or pattern and clinical outcome, although highly exploratory subset analyses suggest potential explanations for this negative finding. Furthermore, neither p53 nor Ki-67 was found to be associated with different clinical outcomes in patients whose tumors express various levels of these markers. Finally, although no treatment interactions were identified with respect to either TS or Ki-67, we found that patients whose tumors stained positively for p53 benefited substantially from the use of adjuvant chemotherapy compared with those who were not treated and that those patients whose tumors were negative for p53 had a substantially worse outcome with the use of 5-FU–based chemotherapy compared with those who were not treated. However, further studies are needed to fully define the role of p53 as a predictor of therapeutic benefit.

APPENDIX
Additional participating institutions and physicians include: Duluth Community Clinical Oncology Program (CCOP), Duluth (James E. Krook, MD); CentraCare Clinic, St Cloud, MN (Harold E. Windschitl, MD); Cedar Rapids Oncology Project CCOP, Cedar Rapids (Martin Wiesenfeld, MD); Siouxland Hematology-Oncology Associates, Sioux City, (John C. Michalak, MD); Iowa Oncology Research Association CCOP, Des Moines, IA (Roscoe F. Morton, MD); Sioux Community Cancer Consortium, Sioux Falls (Loren K. Tschetter, MD); Rapid City Regional Oncology Group, Rapid City, SD (Larry P. Ebbert, MD); Toledo Community Hospital Oncology Program CCOP, Toledo, OH (Paul L. Schaefer, MD), Medcenter One Health Systems, Bismarck, ND (Ferdinand Addo, MD); Ann Arbor Regional CCOP, Ann Arbor, MI (Philip J. Stella, MD); MissouriValley Cancer Consortium, Omaha, NE (James A. Mailliard); and Ochsner CCOP, New Orleans, LA (Carl G. Kardinal, MD).

	ACKNOWLEDGMENTS

 
Supported in part by Public Health Service grant nos. CA-25224, CA-37404, CA-15083, CA-37417, CA-35113, CA-35269, CA-52352, CA-35103, CA-35415, CA-35195, CA-35101, CA-63848, CA-63849, and CA-35272.

	NOTES

 
This study was conducted as a collaborative trial of the North Central Cancer Treatment Group and the Mayo Clinic.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted August 13, 2001; accepted December 5, 2001.

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