Radiol Oncol 1998; 32(4): 407-92.
Phase II study of fluorouracil, leucovorin and interferon alpha-2a in patients with advanced colon cancer
Borut Stabuc, Aleksandra Markovic, Erik Brecelj, Samir Beslija, Tomaz-Edvard Cizej
Institute of Oncology, Ljubljana, Slovenia
Based on in vitro studies that have demonstrated synergy between fluorouracil (5-FU) and leucovorin (LV) as well as between 5-FU and recombinant alpha-2a interferon (IFN) against colon cancer cell lines a phase
II study was carried out to evaluate the toxicity and clinical activity of 5-FU modulated with LV and IFN in patients with metastatic colon cancer. Twenty-two chemotherapy naive patients with measurable metastases of colon cancer have been treated with daily doses of 5-FU 600 mg/m2 in 6-hr intravenous infusion, and of LV 20 mg/m2 intravenously and IFN 6 MU subcutaneously, far 5 days every 4 weeks. Median age was 60 years, median PS (ECOG) was 1 (range 0-2). Liver, soft tissue and lung metastases were found in 12, 5 and 8 patients, respectively. Nineteen patients had a single metastatic site, two double, whereas one had more than two metastatic sites. Patients had 2-9 (mean 5) cycles of treatment. Objective response was observed in 7 patients (32%), and stable disease in 7 patients (32 %). Overall median survival was 12.5 months, and far responders 14.4 months. Responses were generally short and median time far progression was 5.5 months. The most frequent adverse reactions were flu-like syndrome (50%), nausea/vomiting (36%), diarrhoea (13%), stomatitis (27%) and leucopenia (13%).
This regimen of 5-FU with LV and IFN administration does not appear to be superior to previously published schedules of 5-FU with IFN ar 5-FU with LV.
Key words: colonic neoplasms-drug therapy; clinical trials phase II; fluorouracil; leucovorin; interferonalpha; neoplasms metastasis
Introduction
Colon cancer is one of the most common malignancies in Slovenia, with more than 400 new cases diagnosed each year. In 1995, the incidence of colon cancer per 100,000 popu-
Correspondence to: Assist. Prof. Borut Štabuc, MD PhD, Department of Medical Oncology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Tel.: +386 61 302 828; Fax:+386 61 302 828.
lation in Slovenia was 22.2 for male and 21.7 for female.1
Despite better surgical treatment and adjuvant chemotherapy, approximately 50% patients develop metastases. Patients with metastatic colon cancer have uniformly poor
prognosis, because there is no effective therapy.
For more than 40 years fluorouracil (5-FU) has been the mainstay in the treatment of
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colorectal cancer. As a single agent or in combinations with other chemotherapeutic agents, the overall response in advanced colorectal cancer is in the range of 17% to 24%, with median survival time of approximately 11 months.2 In an attempt to improve these results several approaches have been adopted. The most promising is the adjunction of a modulatory agent, leucovorin (LV), which interferes with the mechanisms of 5-FU cytotoxicity. Experimental studies have demonstrated that LV is metabolised to a compound that stabilised the ternary complex formed between the 5-FU metabolite fluo-rodeoxyuridilate and enzyme thymidylate synthase, a key enzyme in DNA synthesis thereby inhibiting enzymatic function and increasing cytotoxicity of 5-FU.3"5 In several randomised trials in metastatic colorectal cancer, 5-FU/LV combination produced statistically significant improvements in response rate compared with 5-FU alone, and a modest improvement in survival.6"8
In preclinical models, combining the recombinant interferon alpha-2a (IFN) and 5-FU resulted in the augmentation of the cyto-toxic effects of 5-FU. The principal mechanism of IFN modulation of 5-FU is currently uncertain. However, evidence exists that this agent alters the 5-FU plasma pharmacokinet-ics by increasing the area under the concentration (AUC)9 and the formation of fluoro-deoxiuridine-monophosphate,10 inhibiting the 5-FU induced upregulation of thymidilate synthase, as well as thymidine kinase,11 and enhancing incorporation of fluorodeoyiuri-dine triphosphate into DNA.12
In 1989, Wadler published promising results of the treatment of metastatic colorec-tal carcinoma with a combination of 5-FU and IFN. The reported response rate was 76% in a subgroup of non-pre-treated patients.13 In 1991, inspired by these results, we started a clinical trial in patients with metastatic colon cancer using double modulation of 5-FU with LV and IFN. The aim of this study
was to evaluate the toxicity, response rate and overall survival in the treatment with this drug combination. However, as the recent reports of studies on 5-FU double modulation by LV and IFN are conflicting,14 we reviewed and discussed available clinical studies using double modulation of 5-FU with LV and IFN.
Patients and methods
Between December 1991 and January 1994, 22 patients with measurable metastatic colon cancer and Eastern Co-operative Oncology Group (ECOG) performances status of O to 2 were entered into the study protocol approved by Medical Ethics Committee at the Ministry of Health of the Republic of Slovenia. The patients who had received no prior chemotherapy for metastatic disease had to meet the following additional criteria: metastatic disease not amenable to surgery, prior adjuvant therapy with 5-FU alone or with levamisol had to be completed >8 months before study entry, no evidence of cardiovascular disease, adequate organ function including normal bone marrow function (leukocyte count >3,500/|L, platelet count >100,000/|L), renal function (creatinin <1.5 mg/dL or creatinin clearance >60 ml/min), and normal hepatic function (bilirubin < 38 mmol/L, liver transaminase level < 3 times the normal values, an albumen value>36 g/L, negative hepatitis B surface antigen).
Patients with active cardiac disease, infection, clinically significant pulmonary and neurologic dysfunction, cerebral metastases, any significant intercurrent illness, requirement of systemic steroids, psychiatric history, or those who had undergone surgery, chemo-immuno- or radiation therapy were not eligible.
In the 4 weeks before entering the trials, the size of the metastatic lesions was determined by computed tomography and/or
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ultrasound and /or X-ray examinations and the serum levels of the carcinoembryonic antigen (CEA) was measured. The size of the lesions was determined after every second treatment cycle. The patients with disease progression had the best supportive care with no additional second line chemotherapy.
Treatment plan
Patients received LV (Leucovorin Ben Venue Laboratories Bedford) 20 mg/m2/day intravenously (i.v.) and half-an hour later 5-FU which was administered in 6-hr continuos intravenous infusion at a dose of 600 mg/m2/day diluted in 1,000 ml of normal saline. 5-FU was obtained commercially. Recombinant human IFN a-2a (Roferon; Hoffman La-Roche) was administered sub-coutaneusly (s.c.) in a dose of 6 MIU consecutively after each 5-FU administrations. Patients received all three drugs for 5 consecutive days. Cycles were repeated at 28-day intervals. The dose of 5-FU was modified for myelosupression, diarrhoea and stomatitis. In grade 3 or 4 toxicity the dose of 5-FU was reduced for 20%. Subsequent courses were delayed until haematological recovery to the following values: granulocytes, 1,500 /¡iL or higher and platelets 100,000 or higher, and resolution of all nonhematologic toxicity to Grade I or baseline. There was no reduction of the doses of LV and IFN.
Response criteria
Treatment response was evaluated after each 2nd cycle. The responders and those with stable disease were subjected to regular clinical and radiological follow up every two months until progression, and then after 3 months until deaths. Tumor response was defined according to World Health Organisation (WHO) criteria.15 A complete response (CR) was defined as the disappearance of all
known disease symptoms on two separate measurements performed in at least 4 week interval; partial response (PR) was defined as a 50% decrease in the sum of products of the largest perpendicular diameters of all measurable lesions for a minimum of 4 weeks, without the appearance of new lesions; stable disease (SD) was defined as a decrease by <50% or increase by <25%, with no new lesions; progressing disease (PD) was defined as a 25% or greater increase in measurable disease or appearance of new lesions. The duration of response was measured for patients with objective response from the onset of objective response to the time of progression. The duration of survival was measured from the first day of treatment until the date of death.
Statistical Analysis
The survival, duration of response and the time to progression were calculated using log-rank test,16'17 while the calculations of the 95% confidence interval for response rate were made by Brookmeyer and Crowley's method.18
Results
The characteristics of the 22 patients entered into this study are listed in Table 1. Seven patients received prior adjuvant therapy consisting of 5-FU and levamisol. The majority of patients had minimal cancer related symptoms. Their ECOG PS was 0-1 except in 1 patient. Patients received 2 to 9 cycles of chemoimmunotherapy (mean 5 cycles). All patients were eligible and evaluable.
Tumour response
The overall response rate was 32% (95% confidence interval (CI) 14% to 52%) (Table 2). All
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Table l. Patients' characteristic
Characteristic	No. of patients Age, years
Median	60
Range	31-78
Male/Female	11/11 ECOG performace status
0/1/2	13/8/1
Prior adjuvant therapy	7
Sites of disease:
-	Liver	12
-	Lung	8
-	Peritoneum	2
-	Lymph nodes	3
No. of metastatic sites
1	19
2	2 3	1
responses were partial. There was no complete response. The median time to achieve initial response was 2.5 months (range 1.5 to 4.0). Most responding patients showed conTable 2. Tumor response
Response
Objective response	7
CR	0
PR	7
Stable disease	7
Progressive disease	8
tinued improvement in their disease status, and the median time to achieve best clinical response was 4.0 months (range 1.5 to S.O). In addition, 32% of patients had stable disease (range 3 to 14 months). Two of twelve patients with metastases involving liver only, two of eight patients involving lung only and two patients with the involvement of both, lung and an extrahepatic site responded. In two patients with lung and soft tissue metastases, CR was observed in one metastatic
site, in the lung of one patient and in the soft tissue of the other. In one patient who had liver, lung and soft tissue metastases CR was observed in the lung and soft tissue whereas in the liver only PR was noticed. The patient who had experienced daily hectic fever to 38.5°C caused by liver metastases, completely resolved the fever within 2 weeks of the initiation of therapy. In the group of seven patients, treated with adjuvant chemotherapy, we did not see any response to chemoim-munotherapy.
Survival
Overall median survival time was 12.5 months and of responders 14.4 months (Figure 1). Median follow up was 12 months. Among 22 patients, 20 patients died. Their survival ranged from 4 to 30 months. Two patients are still alive 26 and 36 months after beginning the study. The median time to progression was 5.5 months in 20 patients. In the patients with objective response the median time to progression was 8.7 months. The two survivors have been without any
Response rate (%) 32 (95% C.I. 14-52%) 0
32 32 36
100
0	6 12 18 24 30 36 42
Survival Time in Months Figure 1. Probability of survival of the treatment group.
No. of patients
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sign of progression in the last 22 and 30 months, respectively.
Toxicity
As shown in Table 3, the side effects of chemotherapy were generally mild and not exceeding the toxicity grade of 3 to 4, neither causing toxicity related deaths nor requiring discontinuation of chemotherapy due to toxicity. In one patient chemotherapy was postponed for 14 days due to grade 2 leucopenia. One patient requested a dose reduction of 5-
thymidylate sinthase. The 5-FU/LV combination produced statistically significant improvements in response rates compared with 5-FU alone in several randomised trials in metastatic colorectal cancer,19-21 although improved survival was noted in two trials only.7'8 Inhibition of thymidine incorporation into DNA is likely to be enhanced by IFN as a result of its inhibitory effect on thymidine kinase.11 Based on this biochemical rationale of inhibiting two key enzymes of 5-FU metabolism, a number of trials have been performed in order to investigate the possibility
Table 3. Toxicity: The worst grade per patient across all cycles
Toxcity grade per patient 2	3
Flu-like	7	4
Nausea/vomiting	6	2
Stomatitis	3	2
Diarrhoea	3	2
Anorexia	3	3
Leukopenia	2	3
Plantar-palmar	5	1
Erythredema		
1
4
FU due to anorexia and plantar-palmar ery-troderma. The most common toxicity was flulike syndrome which occurred in 11 patients. After the 3rd cycle, the majority of the patients did not complain anymore of any of these types of toxicity. Nausea and vomiting grade 1 and 2, mild diarrhoea, leucopenia and plantar-palmar erythroderma occured in 8,3,3, and 6 patients, respectively.
Discussion
In an effort to improve its antitumor activity, a number of strategies to modulate the cyto-toxicity of 5-FU have been investigated. One approach has focused on the use of the LV in combination with 5-FU to stabilise the binding of the active metabolite fluorodeoxyuri-dine monophosphate to its target enzyme,
of biochemical double modulation and to assess the feasibility of this combination (Table 4).
We have demonstrated the feasibility of administering 6 MIU IFN with 5-FU and low dose LV. Our combination of 5-FU/LV/IFN has activity in previously untreated patients with colon cancer. We have observed a 32% overall response rate. The reason for such a response rate and low toxicity may lie in a relatively high dose of 5-FU and the administration of 5-FU in prolonged infusion. A dose - response relationship has been suggested for 5-FU in colon cancer. 5-FU is a drug with very short plasma half-life of approximately 12-18 minutes. The drug has cytotoxic activity against cells in S phase. With bolus administration, a small proportion of cancer cells would be susceptible.22'23 Thus, there exists a sound rationale for the preference of continu-
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Table 4. Results of trials of 5-fluorouracil (5-FU), leucovorin (LV) and interferon alpha (IFN) for treatment of meatastatic colorectal cancer
Reference	5- FU/LV/ IFN regimen	No. of patients	Objective response (%)
Yalavarthi27	5-FU 370 mglm2 i.v. d 2 - 6q 21 d LV 500 mg/m2 i.v. d 2 - 6 q 21 5 MIU INF s.c. d 1 -7 q 21 d	31	23
Seymour28	5FU 400 mg/m2 bolus, then 5FU 400 mg/m2 by i.v. infusion over 22 hours d 1.2 q 14 d LV 200 mglm2 i.v. 6 MIU IFN s.c. every 48 hours troughout	128	31
Recchia29	5-FU370mg/m2 i.v. d 2 -6 q 21 d LV 500 mglm2 i.v. d 2 - 6 q 21 d 3 MIU IFN x 3 s.c. d q 28 d	32	22
Grem30	5-FU 370 mg/m2 i.v. d 2- 6q 21 d LV 500 mglm2 i.v. d 2 - 6 q 21 d 5 MIU IFN s.c. d 1 - 7 q 21 d	54	44
Labianca31	5-FU 400 mg/m2 i.v. x 5 d q 28 d LV 200 mg/m2 i.v. x 5 d q 28 d 3 MIU IFN - a2b s.c. x 5 d q 21 d	36	22
Kreuser32	5-FU 500 mg/m2 i.v. x 7 d q 21 d LV 200 mg/m2 i.v. x 7 d q 21 d 5 MIU IFN - a2b s.c. x 7 d q 21 d	62	29
Cascinu33	5-FU 370 mg/m2 i.v. d 2 - 6 q 21 d LV 200 mg/m2 i.v. d 2 - 6 q 21 d 3 MIU IFN s.c. dl -7 q 21 d	45	51
ous infusion 5-FU to bolus injections. In many trials, objective response rate was higher and toxicity much lower in the patients who received continuous infusion of 5-FU than in the patients treated by bolus injections. 24,25
Numerous trials have been performed with different doses of LV (15 to 500 mg/m2). In general, in clinical trials no difference was indicated in the activity between low (20 mg/m2) and higher doses of LV (250 mg/m2).19,26
The first clinical results of the 5-FU/LV/IFN combination were published in 1990.27 The treatment schedule consisted of 1-20 MIU IFN/day for 5 days and 370-425 mg/m2/day 5-FU, and 500 mg/m2/day LV, given in 5 consecutive days. Only 7 of 31 patients (23%) demonstrated PR, which was disappointing. In the next three years various modes of drug administration were studied in order to define an optimal therapeutic approach (Table 4). Although the majority of phase II studies with 5-FU, LV and IFN have
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demonstrated objective response in approximately 30% of patients with advanced colorectal cancer, their relevance seems hampered by small numbers of patients.
Despite some clinical trials which showed that 5-FU plus IFN is more effective than 5-FU alone in terms of response rate and event free survival, but not of overall survival,34 in three recent randomised clinical trials using double modulation of 5-FU, the investigators found that no benefit could be achieved with the addition of IFN. An Italian trial was aimed to clarify whether IFN could further enhance the therapeutic potential of 5-FU in combination with LV. Eighty-three patients were entered into this trial. The therapy in the first treatment arm consisted of 200 mg/m2/day LV, followed by 370 mg/m2/day 5-FU on days 1-5. In the second treatment arm, 3MIU IFN was administered three times a week subcoutaneusly. The response rates in the 5-FU/LV and IFN arms was 45% and 22%, respectively.29 Pensel randomised 55 patients between treatment with 5-FU/LV with or without IFN administered at a daily dose of 5MIU. Preliminary results did not suggest any statistically significant difference in terms of treatment outcome between the two arms.35 Disappointing results were also reported by the Hellenic Co-operative Oncology Group. Among 95 patients enrolled in trial the response rates in the arm without IFN and in the arm with IFN were 19% and 6%.36
According to phase II studies and preliminary results of three randomised trials, the concept of 5-FU double modulation using IFN and LV does not seem to fulfil its promise either. Therefore, for cost considerations and because combined treatment with 5-FU, LV and IFN seems to result in enhanced toxicity, currently used dose regimens of this approach cannot be recommended for routine use. As the modulatory effects of IFN on chemotherapy in experimental models are highly dependent on the
timing and the dose of IFN, alternative
schedules of 5-FU and IFN combination are
worth exploring in clinical trials.
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