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© 2001 American Society for Clinical Oncology Phase I Clinical and Pharmacogenetic Trial of Irinotecan and Raltitrexed Administered Every 21 Days to Patients With CancerByFrom the Developmental Therapeutics Program, University of Pennsylvania; Center for Clinical Epidemiology and Biostatistics; and Department of Pharmacology, and Center for Pharmacogenetics, Philadelphia, PA. Address reprint requests to James P. Stevenson, MD, 51 N. 39th St, Medical Arts Bldg, Ste 103, Philadelphia, PA 19104; email: james.stevenson{at}uphs.upenn.edu
PURPOSE: Irinotecan and raltitrexed display schedule-dependent synergy in vitro, which supports the clinical investigation of the combination. Functional polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene result in intracellular redistribution of folate derivatives, which may affect raltitrexed-associated cytotoxicity. PATIENTS AND METHODS: Patients with a range of solid cancers and good performance status received irinotecan as a 90-minute infusion on day 1 and raltitrexed as a 15-minute infusion on day 2, repeated every 21 days. Samples were collected for MTHFR C677T genotyping and fasting plasma homocysteine during the first cycle. RESULTS: Thirty-nine assessable patients received 127 cycles of therapy. Irinotecan doses ranged from 100 to 350 mg/m2, and raltitrexed, 1.0 to 4.0 mg/m2. Raltitrexed doses of more than 3.0 mg/m2 were not tolerated and were associated with dose-limiting asthenia, diarrhea, and AST/ALT elevation. Irinotecan/raltitrexed doses of 350/3.0 mg/m2 were well-tolerated; principal toxicities included neutropenia, diarrhea, and fatigue. Two partial responses were observed in patients with pretreated gastroesophageal cancers. Homozygotes with the MTHFR 677 TT polymorphism incurred significantly less raltitrexed-associated toxicity than those with either wild-type or heterozygous genotypes (P = .05). No significant differences were noted in plasma homocysteine values between the genotypic subtypes, and plasma homocysteine levels did not predict the risk of toxicity. CONCLUSION: Irinotecan and raltitrexed doses of 350 and 3.0 mg/m2 are recommended for further study on a day 1, 2 schedule every 21 days. Efficacy results suggest that trials in upper and lower gastrointestinal malignancies are warranted. MTHFR C677T genotypes may be predictive of clinical raltitrexed toxicity.
SINCE ITS APPROVAL for use in metastatic colorectal cancer in 1996, the topoisomerase I inhibitor irinotecan has proven valuable in the treatment of cancer1 because its broad antitumor activity also includes efficacy against lung, upper gastrointestinal tract, gynecologic, and CNS cancers.2-5 The in vitro evidence of synergy between irinotecan and thymidylate synthase (TS) inhibitors such as fluorouracil and raltitrexed has led to clinical investigations of combination regimens.6,7 Saltz et al8 first demonstrated the safety and efficacy of the weekly irinotecan/fluorouracil/leucovorin regimen in phase I/II trials, and both this group and that of Douillard et al9 have recently shown superior progression-free and overall survival for the combination compared with fluorouracil/leucovorin alone in a randomized trial involving previously untreated patients with metastatic colorectal cancer. The quinazoline antifolate raltitrexed is a potent and specific inhibitor of TS, with activity comparable to that of fluorouracil in colorectal cancer and an improved quality of life profile.10 Irinotecan and raltitrexed demonstrate schedule-dependent synergy when applied to colon carcinoma cell lines in vitro: irinotecan followed by raltitrexed 24 hours later resulted in maximum cell kill, although the reverse sequence was found to be additive or antagonistic.7,11 Potentiation of this effect was maximal when greater relative concentrations of raltitrexed were used.11 The combination of irinotecan and raltitrexed is, therefore, of great clinical interest, notably in colorectal cancer, and prompted the trial described here. Grem et al12 reported asthenia, neutropenia, and elevation of hepatic transaminase to be the principal toxicities associated with raltitrexed in their phase I trial and found that doses greater that 4.0 mg/m2 produce prohibitive toxicity. The clinical physiologic and molecular determinants of toxicity in patients receiving folate-based TS inhibitors such as raltitrexed are unknown.13 Given the requirements of raltitrexed for (1) the reduced folate carrier for intracellular entry, (2) polyglutamation by folylpolyglutamate synthetase for cellular retention and maximal inhibition of TS, and (3) its competition with 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) for TS binding, it is quite plausible that differences in individual folate status may contribute to observed toxicity and possibly to efficacy in patients receiving therapy. Data lending support to this hypothesis have been presented by Zervos et al.14 They found that folate-deficient patients (as indicated by elevations of plasma homocysteine and cystathionine) experienced greater toxicities during therapy with the multitargeted antifolate LY231514, an inhibitor not only of TS but also of the folate-dependent enzymes dihydrofolate reductase and glycinamide ribonucleotide formyltransferases. There are multiple determinants of functional folate status in humans. The major components of the folate/homocysteine axis are shown in Fig 1. A key regulatory step in this pathway involves the enzyme methylenetetrahydrofolate reductase (MTHFR), which catalyzes the formation of 5-methyltetrahydrofolate from 5,10-methyleneTHF.15 5-methylTHF functions as a methyl donor in the methionine synthase-mediated conversion of homocysteine to methionine, although 5,10-methyleneTHF is critical to the generation of purine and pyrimidine nucleosides, most notably as a cofactor in the formation of thymidylate from deoxyuridine monophosphate via TS.
Although folate conservation by the human body is great and the Western diet is generally folate-replete, functional polymorphisms of enzymes involved in the folate/homocysteine axis may affect the cellular distribution of folate derivatives. In 1988, Kang et al16 identified a form of MTHFR in almost 5% of whites that is biochemically thermolabile in that its residual activity after incubation at 45°C in vitro is significantly less than that of the wild-type enzyme. After the cloning of MTHFR cDNA, the mutation underlying the thermolabile isoform was identified as a C  T transition at nucleotide 677 that mandates the substitution of an evolutionarily conserved alanine at residue 222 by a valine.17,18 This mutation is panethnic, and several studies in different countries have established that individuals with the 677 homozygous for the polymorphism (TT) MTHFR genotype are at increased risk of being hyperhomocysteinemic, particularly when their plasma folate levels are below the population median.19-22 TT individuals have also been reported to have lower circulating 5-methyTHF concentrations and accumulate formulated tetrahydrofolate polyglutamates (ie, 5,10-methenylTHF) in RBCs.21,23 The implications of this functional MTHFR variant for therapy with folate analogs that inhibit TS are readily apparent: the increased availability of 5,10-methyleneTHF as a result of impaired MTHFR activity could compete with drugs like raltitrexed for polyglutamation by folylpolyglutamate synthetase and binding to TS, diminishing cytotoxicity, and observed clinical toxicity and efficacy. We, therefore, sought to study MTHFR C677T genotypes in patients with advanced cancer who received raltitrexed as part of a phase I trial in combination with irinotecan.
Patient Population Patients were enrolled onto this trial between February 1997 and July 1999. Patients at least 18 years of age with a histologic diagnosis of cancer with no effective treatment options were considered eligible. An Eastern Cooperative Oncology Group performance status  2 and life expectancy  12 weeks were required. All patients had recovered from previous treatments, had measurable or assessable disease, and had received no therapy 28 days before screening. Eligibility required adequate bone marrow function (neutrophils 1,500/mm3; platelets 100,000/mm3), serum creatinine 1.5 mg/dL, total bilirubin less than 2.0 mg/dL, and ALT and AST 5 times the upper normal level. All patients received information on the purpose and conduct of this study and provided written informed consent in accordance with federal, state, and institutional guidelines. Pretreatment evaluation consisted of a history and physical examination, complete blood count, serum chemistries, electrolytes and creatinine, prothrombin time and activated partial thromboplastin time, urinalysis, ECG, and assessment of Eastern Cooperative Oncology Group performance status. Fasting plasma homocysteine values were obtained on day 1 before treatment. Blood counts and biochemical profiles were performed twice weekly during the first cycle of treatment, then once weekly thereafter. Toxicity during each cycle was assigned according to the Cancer Therapy Evaluation Program common toxicity criteria (CTC), version 2.0. Lesions noted at baseline that were measured or evaluated by radiographic scan or x-ray were reviewed after every third treatment course and evaluated for response according to standard criteria.24
Drug Administration An IV antiemetic regimen of dexamethasone 10 mg to 20 mg and ondansetron 24 mg to 32 mg or granisetron 10 µg/kg was administered 30 minutes before each dose of irinotecan. Prochlorperazine was not used as an antiemetic on the day of irinotecan dosing because of its association with akathisia. Atropine 0.25 mg to 1.0 mg was administered by IV before irinotecan to prevent the early cholinergic syndrome. Loperamide was provided to all patients at the start of therapy for the management of late diarrhea. The patients were instructed to ingest 4 mg followed by 2 mg every 2 hours until there had been no diarrhea for at least 12 hours. Growth factor support was not routinely used but was left to the discretion of the treating physician.
Study Design
Genotyping for MTHFR
A total of 41 patients were entered onto this study. Thiry-nine were assessable for toxicity. Two patients only received day one of therapy: one patient developed rapid disease progression, and another patient did not return for further treatment. The 39 assessable patients received 127 cycles of irinotecan and raltitrexed. The demographic characteristics of those enrolled are listed in Table 1. There are no patients who remain on the study.
Toxicity The combination was generally well-tolerated by patients until irinotecan and raltitrexed doses of 225 mg/m2 and 4.0 mg/m2 were explored (Table 2). At these dose levels, DLT in the form of grade 4 diarrhea and grade 3 elevation of hepatic transaminases were encountered in two of the first three patients treated. At that point, raltitrexed dose was decreased to 3.0 mg/m2 and irinotecan escalation continued. With this schema, irinotecan was escalated to 350 mg/m2, representing the standard single-agent dose on a 3-week schedule. Two episodes of grade 4 neutropenia occurred at these doses (irinotecan 350 mg/m2 and raltitrexed 3.0 mg/m2) but they were short-lived (< 5 days) and not complicated by fever or infection. Other toxicities included diarrhea, nausea, alopecia, and fatigue, but these were not dose-limiting. We, therefore, found doses of irinotecan 350 mg/m2 and raltitrexed 3.0 mg/m2 to be suitable for further study on this schedule.
MTHFR Genotype and Toxicity MTHFR C677T genotyping was performed using PMNs from 33 of 39 assessable patients. Six patients were not informative as a result of sample inadequacy. Informative samples were represented across all dose levels (Table 3). The patients with informative samples were all white. Twenty-six of 33 patients were found to be CC or CT for the MTHFR 677 C T transition, although seven (21%) of 33 were TT. When toxicities typically associated with raltitrexed were examined separately in the genotyped patients, TT homozygotes had a lesser incidence of grade 3/4 neutropenia (14% v 27%; P = .33), grade 3/4 asthenia (0% v 27%; P = .15) and all grades of hepatic transaminase elevation (28% v 44%; P = .25) compared with CT/CC genotype patients receiving therapy (Table 4). When all instances of raltitrexed-associated toxicities were compared between CC/CT patients and TT patients, the difference was found to be significant (14% v 33%; P = .05).
MTHFR Genotype and Plasma Homocysteine Fasting plasma homocysteine values were measured in genotyped patients. The mean homocysteine level for CC/CT patients was 9.3 µmol/L (range, 5.1 µmol/L to 16.9 µmol/L) and for TT homozygotes was 8.1 µmol/L (range, 5.9 µmol/L to 11.7 µmol/L; P = .39). Only two patients had values greater than our institution’s upper limit of normal of 12.0 µmol/L, both with wild-type CC genotypes. Thus there was no suggestion that TT individuals in this study were hyperhomocysteinemic. There also was no relationship observed between individual homocysteine values and toxicity.
Efficacy
Clinical trials of irinotecan in combination with inhibitors of TS (including fluorouracil) have confirmed the preclinical synergy observed with their sequential application to human cancer cell lines. The irinotecan/fluorouracil/leucovorin regimen has recently been shown to produce superior survival times (14.8 months v 12.6 months) than either irinotecan or fluorouracil alone as front-line therapy for metastatic colorectal cancer.9 However, the combination is toxic, and nearly one quarter of treated patients will experience grade 3/4 diarrhea or grade 4 neutropenia. The development of potentially less toxic schedules of administration, alternative combinations, and markers that may identify suitable candidates for therapy is therefore of interest. We found the combination of irinotecan and raltitrexed to be both active and well-tolerated when administered sequentially on days 1 and 2 every 3 weeks in this pretreated patient population. The standard single-agent doses of both agents were tolerable on this schedule, without evidence for overlapping toxicity, and doses of 350 mg/m2 and 3.0 mg/m2 for irinotecan and raltitrexed, respectively, are recommended for further study. The incidence of diarrhea was not excessive, with no grade 4 episodes noted at the final dose level. Grade 4 neutropenia was noted to occur. However, these episodes were generally of short duration and without clinical sequelae. Nausea, asthenia, and hepatic transaminitis were the other principal toxicities observed with the combination. These side effects did not limit long-term administration to patients who were benefiting from therapy. Five patients received six cycles or more, including one woman with colorectal cancer who was treated with 14 cycles over a period of 10 months. Ford et al27 recently reported the results of their phase I trial of irinotecan and raltitrexed in patients with advanced cancers of the gastrointestinal tract. The schedule of administration differed slightly in that both drugs were given on day one every 21 days (irinotecan followed by raltitrexed). They also recommended doses of 350 mg/m2 and 3.0 mg/m2 for further evaluation, and observed a similar side effect profile to that seen in this study. Partial responses were reported in six patients with colorectal cancer, and no pharmacokinetic interaction was observed between the two drugs. Although we do not report pharmacokinetics here, we also would not expect a pharmacokinetic interaction, given our two-day schedule of administration. The activity, tolerability, and ease of administration of both drugs shown in these reports suggest that further trials of irinotecan and raltitrexed in gastrointestinal cancer patients are warranted. With the clinical development of more active and targeted chemotherapeutic agents, in the future oncologists will likely have an assortment of choices for therapy. The identification and selection of patients appropriate for a specific treatment will, therefore, be crucial. An extreme example of the need to identify such associations can be found in patients with dihydropyrimidine dehydrogenase deficiency who experience severe and life-threatening toxicity when given fluorouracil.28 Our results suggest that MTHFR C677T genotype may have predictive value in patients receiving raltitrexed. As hypothesized, TT homozygote patients experienced less raltitrexed-attributable toxicity than CC/CT patients did. When looked at as a whole, this difference was significant. The partial response rate and number of patients in this study were too small to explore any association between MTHFR C677T genotype and clinical efficacy. However this would be of interest in a larger study of the combination as first-line therapy, as well as in other trials of folate-based TS inhibitors. The accumulation of formulated THFs resulting from TT homozygosity would be expected to have a reciprocal effect on toxicity in patients treated with fluorouracil, as fluorouracil cytotoxicity via 5-FdUMP-TS ternary complex formation is potentiated by the sequential binding of 5,10-methyleneTHF.29 Recent clinical data lends support to this alternative association, as Toffoli et al30,31 observed greater toxicity (neutropenia) in TT homozygotes receiving adjuvant cyclophosphamide, methotrexate, and fluorouracil for breast cancer than CC/CT genotypes. The implications of simple alterations in the intracellular distribution of folate derivatives resulting from reduced MTHFR activity would theoretically be of great consequence, producing effects on DNA repair, methylation, and gene expression.32,33 Accordingly, the MTHFR C677T polymorphism has been related to carcinogenesis. An analysis from the Physicians’ Health Study revealed that folate-replete men with the TT genotype had a three-fold reduction in colorectal cancer risk compared with CC/CT genotypes.34 TT homozygosity was also found to protect against acute lymphocytic leukemia, and an association with endometrial cancer has been postulated.35,36 Separate reports have described the loss of heterozygosity of the MTHFR gene in ovarian and colon cancers, suggesting that acquired loss of function of MTHFR may play a role in tumorigenesis.37,38 Our findings lend support to the growing body of data regarding the links between folate status, cancer, and response to chemotherapy. Further pharmacogenetic investigation of additional polymorphisms in MTHFR as well as other enzymes in the folate/homocysteine axis may lead to the establishment of objective genetic screens for patients to identify those who are more likely to benefit from specific therapies such as raltitrexed.
Supported by a grant from the Margaret Q. Landenberger Foundation, Philadelphia, PA.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
