Schedule-selective biochemical modulation of 5-fluorouracil in advanced colorectal cancer – a phase II study
© Tomlinson et al; licensee BioMed Central Ltd. 2002
Received: 21 November 2001
Accepted: 2 May 2002
Published: 2 May 2002
5-fluorouracil remains the standard therapy for patients with advanced/metastatic colorectal cancer. Pre-clinical studies have demonstrated the biological modulation of 5-fluorouracil by methotrexate and leucovorin. This phase II study was initiated to determine the activity and toxicity of sequential methotrexate – leucovorin and 5-fluorouracil chemotherapy in patients with advanced colorectal cancer.
Ninety-seven patients with metastatic colorectal cancer were enrolled onto the study. Methotrexate – 30 mg/m2 was administered every 6 hours for 6 doses followed by a 2 hour infusion of LV – 500 mg/m2. Midway through the leucovorin infusion, patients received 5-fluorouracil – 600 mg/m2. This constituted a cycle of therapy and was repeated every 2 weeks until progression.
The median age was 64 yrs (34–84) and the Eastern Cooperative Group Oncology performance score was 0 in 37%, 1 in 55% and 2 in 8% of patients. Partial and complete responses were seen in 31% of patients with a median duration of response of 6.4 months. The overall median survival was 13.0 months. The estimated 1-year survival was 53.7%. Grade III and IV toxic effects were modest and included mucositis, nausea and vomiting.
This phase II study supports previously reported data demonstrating the modest clinical benefit of 5-FU modulation utilizing methotrexate and leucovorin in patients with metastatic colorectal cancer. Ongoing studies evaluating 5-fluorouracil modulation with more novel agents (Irinotecan and/or oxaliplatin) are in progress and may prove encouraging.
Although a number of recently introduced chemotherapeutic agents have demonstrated significant anti-tumor activity in advanced colorectal cancer, [1–9] standard therapy for patients with metastatic disease remains 5-fluorouracil (5-FU) – based chemotherapy. A vast series of pre-clinical and clinical studies have suggested 5-FU is a more active anti-tumor agent when modulated by a host of compounds including leucovorin, [11–13] methotrexate, folinic acid, [15, 16] N-phosphonacetyl-L-aspartic acid (PALA) [17–22] and recombinant interferon alfa-2a (IFNaα-2a). [23, 24] Protracted venous infusion (PVI) 5-FU regimens have also resulted in increased response rates in some studies, but only results in a modest benefit in median survival. [25, 26] Oral fluoropyrimidines and oral regimens using prodrugs of 5-FU or inhibitors of dihydropyrimidine dehydrogenase (DPD) which pharmacologically simulate the intravenous continuous infusion administration of 5-FU are currently under clinical evaluation. Although these studies are important and may demonstrate equivalency, it is not clear these oral compounds will result in a significant improvement compared to the intravenous infusion of 5-FU.
Potentiation of the anti-tumor activity of 5-FU by methotrexate (MTX) and leucovorin (LV) requires careful scheduling to achieve the most favorable interactions between these 3 drugs. In vitro and in vivo studies have both suggested a synergistic as well as antagonistic effect when 5-FU, MTX and LV are used in combination. [28–32] In this report, two modulators of 5-FU were used in a sequential manner, anticipating an enhancement of the efficacy of 5-FU before the development of 5-FU resistance and subsequent disease progression. The order of administration of the three agents was chosen to optimally exploit the reported differing mechanisms of biochemical modulation. The objectives of this phase II study were to determine the activity and toxicity of MTX/LV/5-FU in patients with advanced colorectal cancer.
Patients and Methods
Between August of 1987 and August of 1988, 97 patients with advanced colorectal cancer were enrolled onto this study. Informed consent was obtained in writing from all patients according to Institutional Review Board guidelines. Eligibility criteria included histologic proof of metastatic colorectal carcinoma and the presence of at least one bidimensionally measurable lesion on computed tomography scan. All patients were required to have an Eastern Cooperative Oncology (ECOG) Group performance score of 0 – 2 and have a life expectancy greater than 2 months. Patients also were required to meet the following criteria: adequate bone marrow, renal, and hepatic function as evidenced by a white blood cell count = 4,000 cells/ul, platelet count = 100,000 cells/ul, creatinine = 2.0 mg/dl or creatinine clearance of 60 ml/min and a serum bilirubin = 1.5 mg/dl and no serious intercurrent medical or mental illness. Patients could not have received prior chemotherapy (excluding adjuvant chemotherapy).
Weekly complete blood counts, including platelet counts and white blood count differentials were conducted to determine the level of myelosuppresion. Before every other cycle of treatment (q 4 weeks), a chest x-ray and complete chemistry panel including electrolytes and serum creatinine were obtained. All lesions were measured bidimensionally and assessed for changes by either an imaging study (computed tomography, chest x-ray, ultrasound) or by clinical examination to determine response to therapy. Treatment was continued until disease progression became evident or side effects became intolerable.
This study was planned to recruit 50 evaluable patients, allowing us to estimate objective tumor response to within +/- 15% with 95% confidence. Additional patients were subsequently accrued to increase the precision of our estimates of response and survival. The trial was conducted with one interim analysis after nine evaluable patients. If no patient had experienced an objective tumor response, the study would have been terminated. The two-stage design used in this study was first proposed by Ed Gehan in 1961 (Gehan E.A. The determination of the number of patients required in a preliminary and a follow-up trial of a new chemotherapeutic agent. J. Chron. Dis. 13:346–353, 1961). The probability of observing no responses was less than 5% if the true response was 30% or greater. Progression-free survival was defined as the time from study entry to the first observation of disease progression or death as a result of any cancer. Survival was defined as the time of study entry until death as a result of any cause. These time-to-event parameters were summarized using Kaplan-Meier product-limit estimates. Log rank tests were used to assess which factors were univariately predictive of the time to progression and survival, and the Cox proportional hazards regression model was used to assess which factors were jointly predictive of these outcomes. The statistical analysis was performed using the SAS package, version 6.12 (SAS Institute, Cary, NC).
Median Age Years (range)
Treatment and Response
Percent of Ideal Dose (mean ± SD)
# of Patients
98.9 ± 7.2
99.9 ± 0.7
98.8 ± 10.3
90.1 ± 18.9
91.8 ± 17.4
86.3 ± 24.4
88.6 ± 19.9
87.8 ± 20.2
84.7 ± 44.2
90.3 ± 19.8
93.5 ± 16.4
89.0 ± 19.7
83.0 ± 24.2
89.7 ± 19.3
84.3 ± 22.9
76.7 ± 28.2
80.7 ± 26.7
77.2 ± 28.7
# of Patients
Grade II (%)
Grade III (%)
Grade IV (%)
Non Hematologic Toxicity
Best Objective Response
# of Patients (%) (n = 90 evaluable)
95% C. I.
5.5 – 19.5
12.3 – 29.8
21.8 – 41.7
26.8 – 47.5
22.8 – 42.9
Subgroup analysis of survival
# of Patients
Median Survival (months)
Log rank p-value
Outcome in relation to sites of metastatic disease
Site of Metastasis
# of Patients
Median Survival (mo.)
p – value
# of Metastasis
The rationale, at least in part, for this phase II study comes from pre-clinical data suggesting MTX and LV potentiate the anti-tumor activity of 5-FU. The cytotoxic effects of 5-FU are mediated through inhibition of DNA and/or RNA synthesis. DNA synthesis inhibition is mediated by the binding of the active 5-FU metabolite 5-fluoro-2'deoxyuridine – 5-monophosphate (F-dUMP) to the enzyme thymidylate synthetase (TS). This binding is enhanced by high concentrations of reduced folate cofactors. Although this ternary complex is covalent, it dissociates with a half-life of 2–3 hours in the absence of excess 5, 10-methylene tetrahydrofolate. Thus high levels of 5, 10-methylene tetrahydrofolate derived from LV not only allows optimum ternary complex formation, but also prevents subsequent breakdown of the complex. Additional in vitro studies suggest cells may need prolonged exposure to reduced folates in order to obtain maximum inhibition of TS by FdUMP. A second observed means of potentiating the cytotoxic effects of 5-FU is through the pre-treatment of cells with MTX. MTX-induced enhancement of 5-phosphoribosyl-1-pyrophosphate (PRPP) pools with subsequent increased synthesis of 5-FU nucleotides and incorporation of 5-fluoriuridine – 5'-triphosphate (FUTP) into RNA has been proposed as the basis for this biochemical modulation. [37–39] Subsequent in vivo studies using fluorine 19 magnetic resonance spectroscopy confirmed a three-fold increase in 5-fluoronucleotide (FNuct), the active 5-FU metabolite, following the pre-treatment of animals with MTX . These observed changes in 5-FU catabolism resulted in a greater anti-tumor activity than MTX given alone or MTX given after 5-FU. Consistent with these pre-clinical studies, the design of this phase II trial involved a fluorouracil infusion that was initiated one hour into the 2 hour high dose infusion of LV and 36 hours after the initial methotrexate dosing.
Our phase II study provides long-term efficacy and safety data for sequential MTX-LV-5-FU chemotherapy administered on a 14-day cycle. The 31% response rate and median survival duration (median 13.0 months) observed in this trial are slightly improved when compared to the results reported for intravenous 5-FU therapy. Overall, treatment with sequential MTX-LV-5-FU was very well-tolerated. Grade III diarrhea was seen in only 8% of patients, grade III and IV mucositis in 8% and 1% of patients respectively and grade III and IV nausea/vomiting in 11% and 1% of patients respectively. The most common hematologic toxicity observed was grade II and III anemia in 18% and 4% of patients respectively.
Other similar studies have shown comparable results in terms of the efficacy and safety of MTX-LV-5-FU for patients with advanced colorectal cancer [44–48]. In a meta analysis of eight randomized trials of 5-FU/MTX versus 5-FU alone, the complete and partial response rates for the 5-FU patient cohort was 10% compared with 19% for patients receiving MTX/5-FU. In addition, the median survival was improved for patients receiving MTX/5-FU, 10.7 months versus 9.1 months for patients receiving 5-FU alone (p = .024). Marsh et al. in a randomized trial, reported a statistical improvement in response, time to progression (9.9 months versus 5.9 months) and median survival (15.3 months versus 11.4 months) when the interval between the MTX and 5-FU infusion was increased from 1 hour to 24 hours.
We suggest our results and the pre-clinical and clinical data discussed indicate the sequence of administration of 5-FU with MTX and LV is important. Future studies combining 5-FU with other cytotoxic / biologic compounds should consider the mechanism of the interaction and incorporate that information into the design of the trial.
Protracted venous infusion
Eastern Cooperative Oncology
5-fluoro-2'deoxyuridine – 5-monophosphate
5-fluoriuridine – 5'-triphosphate
The authors wish to thank Carrie G. Blackstock, Ph.D., UNC – Chapel Hill Department of English, for her editorial support.
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