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Clinical Cancer Research Vol. 8, 2488-2498, August 2002
© 2002 American Association for Cancer Research

Clinical Trials

Modulation of the Fas Signaling Pathway by IFN-{gamma} in Therapy of Colon Cancer: Phase I Trial and Correlative Studies of IFN-{gamma}, 5-Fluorouracil, and Leucovorin1

Lee S. Schwartzberg, Istvan Petak, Clinton Stewart, P. Kellie Turner, Jeri Ashley, David M. Tillman, Leslie Douglas, Ming Tan, Catherine Billups, Rudolf Mihalik, Alva Weir, Kurt Tauer, Steve Shope and Janet A. Houghton2

The West Clinic, Memphis, Tennessee 38120 [L. S. S., J. A., A. W., K. T., S. S.]; Division of Molecular Therapeutics, Department of Hematology-Oncology [I. P., D. M. T., L. D., J. A. H.], Department of Pharmaceutical Sciences [C. S., P. K. T.], and Department of Biostatistics [M. T., C. B.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; and First Institute of Pathology and Experimental Cancer Research, Semmelweiss University of Medicine, Budapest, Hungary 1085 [R. M.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Patients and Methods
 Results
 Discussion
 REFERENCES
 
Potentiation of 5-fluorouracil/leucovorin (FUra/LV) cytotoxicity by IFN-{gamma} in colon carcinoma cells is dependent on FUra-induced DNA damage, the Fas death receptor, and independent of p53 and RNA-mediated FUra toxicity, which occurs in normal gastrointestinal tissues. This provides a rationale for enhancing the selective action of FUra/LV by IFN-{gamma} in the treatment of colorectal carcinoma.

Based on results from our preclinical studies we designed a Phase I trial combining FUra (370 mg/m2) and LV (200 mg/m2), i.v. bolus daily x 5 days, with escalating doses of IFN-{gamma} (10–100 µg/m2) s.c. on days 1, 3, and 5, every 28 days. Twenty-five patients with carcinomas were enrolled; 6 patients received IFN-{gamma} on days 1 and 3 only.

The dose-limiting toxicity, stomatitis, occurred most frequently at 100 µg/m2 IFN-{gamma}. Minor response or SD was observed in 2 of 9 patients and in 4 of 12 patients at dose levels of <=50 µg/m2 and >=75 µg/m2 IFN-{gamma}, respectively. Three evaluable chemonaive patients demonstrated partial response (2) or complete response (1). Serial plasma samples revealed peak FUra concentrations of >100 µM; at 100 µg/m2 IFN-{gamma} plasma concentrations >5 units/ml persisted for 6.5 h and >1 unit/ml for 28.5 h. The pharmacokinetic parameters of IFN-{gamma} correlated with a 2–3-fold up-regulation of Fas expression at 24 h in CD15+ cells in peripheral blood samples. Furthermore, clinically relevant IFN-{gamma} concentrations up-regulated Fas expression and sensitized HT29 colon carcinoma cells in vitro to FUra/LV cytotoxicity.

On the basis of the modulation of Fas signaling, FUra/LV combined with IFN-{gamma} has shown activity in a Phase I trial in colorectal carcinoma and warrants additional evaluation in Phase II.


   Introduction
Top
 ABSTRACT
 Introduction
Patients and Methods
 Results
 Discussion
 REFERENCES
 
FUra3 remains the cornerstone of treatment for patients with metastatic colorectal cancer and other GI malignancies. Optimal use of FUra depends on biochemical modulation of its antineoplastic effect. LV, a reduced folate, enhances the effect of FUra on the cellular target, the enzyme thymidylate synthase, thereby potentiating cellular sensitivity and enhancing response rates (1, 2, 3, 4, 5) . Recent trials have added new drugs such as the topoisomerase I inhibitor irinotecan to FUra/LV combinations. The three-drug combination leads to a modest increase in overall survival at the cost of significant increase in morbidity and early mortality (6 , 7) . Other creative strategies, including rationally designed modulation of differential gene expression based on specific cellular characteristics in colon cancers, are urgently needed.

It is well known that FUra induces cytotoxicity by two predominant mechanisms: (a) inhibition of thymidylate synthase, which is potentiated by LV through elevating pools of reduced folates; DNA damage subsequently occurs, and tumor cells die by the process of thymineless death (8, 9, 10) ; and (b) incorporation into RNA, which results in aberrant processing of RNA species, has been associated with GI toxicity observed with FUra in preclinical models (11 , 12) , and provides a rationale for the selective action of FUra/LV in the treatment of colon cancer.

The cell surface receptor Fas (APO-1; CD95) is a member of the tumor necrosis factor receptor family of death receptors, which induce apoptosis in sensitive cells on binding to their specific death ligands. Fas and its ligand FasL are known regulators of apoptosis in cells of the immune system (13) . Analyses of tissues from mice demonstrated expression of Fas in tissues largely characterized by high rates of cell turnover and apoptotic cell death, including epithelial tissues (14) . Fas is highly expressed in normal human colonic epithelial cells, and its expression is progressively decreased during tumor progression from normal epithelium to adenocarcinoma in ~50% of the cases (15 , 16) . Several reports have now confirmed activation of the Fas signaling pathway in the mechanism of FUra action both in vitro (9 , 17) and in vivo (18) .

Thymineless death induced by thymidylate synthase inhibition from FUra/LV occurs downstream of DNA damage by signaling via the Fas (CD95/APO-1) death receptor (19 , 20) . Binding of the natural ligand FasL or a cytolytic anti-Fas antibody leads to a cascade of downstream events including induction of the death-inducing signaling complex and generation of active caspases, which induce apoptosis. The Fas-dependent component of FUra-induced cytotoxicity has been demonstrated in colorectal carcinoma cell lines, hepatocarcinoma, and other malignant cells (9 , 17 , 18) . Sensitivity to Fas-mediated apoptosis has correlated with the level of Fas expressed in human colon carcinoma cell lines (20) .

Based on preclinical studies conducted in our laboratories (9) , we developed a rationale for increasing the selectivity and antitumor action of FUra combined with LV in the therapy of colorectal carcinoma based on: (a) IFN-{gamma}-induced up-regulation of Fas expression; and (b) IFN-{gamma}-induced sensitization to FUra/LV in a Fas-dependent manner in human colon carcinoma cell lines. This rationale is depicted in Fig. 1Citation . We have shown previously that in human colon carcinoma cell lines demonstrating FUra/LV-induced DNA damage, the cytotoxic action of FUra/LV was reversed by dTHd and was significantly potentiated by IFN-{gamma} (HT29, GC3/c1, and VRC5/c1). This is in contrast to cell lines that demonstrate RNA-mediated FUra cytotoxicity, as would be anticipated in normal GI tissues where cytotoxicity was not influenced by IFN-{gamma} treatment (HCT8 and HCT116). These data suggested that sensitization of colon carcinomas to FUra/LV by IFN-{gamma}-induced modulation of Fas signaling would be greater than potentiation of toxicity to normal GI tissues, thereby increasing the potential for an enhanced therapeutic advantage. Furthermore, IFN-{gamma}-induced sensitization of FUra/LV cytotoxicity occurred in cell lines that expressed mutant p53 alleles (HT29, GC3/c1, and VRC5/c1) and, therefore, was independent of the function of a wild type p53 gene. This is also of importance because >75% of colon carcinomas express predominantly mp53 (21) . In addition, the requirement for Fas expression in sensitization to FUra/LV cytotoxicity by IFN-{gamma} was demonstrated in Caco2 cells, where FUra/LV induced DNA damage (dTHd reversible) but in the absence of Fas expression were not sensitized by cotreatment with IFN-{gamma} (9) .



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  		Fig. 1. Rationale: potentiation by IFN-{gamma} in human colon carcinoma cells is dependent on FUra-induced DNA damage and the Fas death receptor, and is independent of p53 and RNA-mediated FUra toxicity, which occurs in normal GI tissues (2) .

 
Based on this rationale, we designed a Phase I trial adding IFN-{gamma} to a standard regimen of FUra/LV. The goals of this study were to determine the safety and tolerability of the combination, evaluate the pharmacokinetics of IFN-{gamma} and FUra, and explore potential interactions. Additionally we sought to determine the ability of IFN-{gamma} at clinically achievable doses to induce Fas up-regulation both in vivo and in vitro.


   Patients and Methods
Top
 ABSTRACT
 Introduction
 Patients and Methods
Results
 Discussion
 REFERENCES
 
In Vitro Studies to Design IFN-{gamma} Dosing Regimen.
Cell surface Fas expression was determined in HT29 human colon carcinoma cells by FACS analysis using a PE-conjugated DX2 anti-Fas mAb (PharMingen) using standard procedures after a 2-h IFN-{gamma} exposure at concentrations from 1 to 10 units/ml, as described previously (22) .

Patients.
Patients with stage IV colorectal, gastric, pancreatic, small intestinal, esophageal, or gallbladder cancer, either chemo-naive or previously treated, were eligible for this trial. Eligibility criteria included age >=18 year; ECOG performance status 0–2 (ambulatory and capable of all self-care); life expectancy of >=12 weeks, no chemotherapy within 4 weeks of treatment (6 weeks for nitrosoureas); adequate hematopoietic (absolute neutrophil count >1000/mm3, hematocrit >27, and platelet count >100,000/mm3), hepatic (bilirubin <2.0 mg/dl, alkaline phosphatase <5x normal, ALT <5x normal), and renal (creatinine <2.0 mg/dl) function; no coexisting medical conditions severe enough to limit compliance with the protocol; no chronic diarrhea >5 bowel movements/day; no prior exposure to IFN-{gamma}; and no active serious infection. All of the patients gave written informed consent according to federal and institutional guidelines before the commencement of treatment.

Patient Evaluation and Follow-Up.
At study entry, all of the patients had a complete medical history and physical examination, complete blood cell count with differential, chem-12 and liver panel, prothrombin time, and ECOG status. The presence of measurable or evaluable disease was evaluated by computed tomography, plain radiograph, and/or palpation within 30 days of study entry. A complete blood cell count and liver panel was obtained weekly through the first 8 weeks and thereafter biweekly until the patient came off study. Measurements of lesions were obtained by restaging with the same imaging techniques before the third, fifth, and seventh cycle of therapy. A patient diary was kept weekly for GI toxicity and use of pain medications. Patients were allowed to continue treatment if they did not develop PD, did not have intolerable toxicity despite dose reductions, or wished to voluntarily withdraw from the study.

A CR was defined as the disappearance of all of the disease on two measurements separated by a minimum of 4 weeks. A PR required more than a 50% reduction in the overall sum of the bidimensional products of all of the measurable lesions determined by two observations not <4 weeks apart. MR was defined as <50% but >25% decrease in the sum of the products of the perpendicular diameters of measurable disease without the appearance of new lesions. PD was defined as at least an increase of 25% in the overall sum of the bidimensional product of measurable lesions compared with baseline or the appearance of new lesions.

Clinical Protocol.
The trial was a dose-escalation study of IFN-{gamma} (Intermune Pharmaceuticals, Palo Alto, CA) added to a standard regimen of FUra (370 mg/m2) and LV (200 mg/m2) administered i.v. daily x 5 days every 28 days according to the Mayo Clinic regimen (1) . IFN-{gamma} was administered on days 1, 3, and 5 s.c. before LV given by a 15-min infusion followed by FUra given as a bolus i.v. injection. Scheduling of IFN-{gamma} on days 1, 3, and 5 was based on preclinical studies that demonstrated a sustained rise and maintenance of cell surface Fas expression after a relatively short exposure of HT29 human colon carcinoma cells to the cytokine. Fig. 2Citation demonstrates that after 2-h exposure of HT29 cells to 1, 3, 5, or 10 units/ml IFN-{gamma}, Fas was elevated by 1.5-fold at 1 unit/ml, by 2-fold at 3 units/ml, and by 3-fold at 5 and 10 units/ml. Elevation of Fas was maximal at 24 h and was followed by a gradual decline. Based on these results, IFN-{gamma} was scheduled on days 1, 3, and 5 in patients to provide sustained elevation in Fas expression throughout the time that FUra and LV were administered. The initial IFN-{gamma} dose was 10 µg/m2 with escalations to 25, 50, 75, and 100 µg/m2 in cohorts of 3 patients until a MTD was determined. The MTD was the highest dosage that induced DLT in <33% of patients. Dose escalation did not occur until all 3 of the patients completed one cycle of therapy (28 days) without DLT. If 1 patient in a cohort exhibited DLT, an additional 3 patients were added to the cohort. When DLT was observed in 2 of 6 patients, dose escalation was terminated and the prior dose was considered the MTD. The National Cancer Institute Common Toxicity Criteria, version 2.0, was used for toxicity grading and adverse reporting. DLT was defined as grade 3 nonhematologic or grade 4 hematologic toxicity. Intraindividual dose reduction by one level was permitted for individuals who experienced DLT. A total of two dose reductions was allowed.



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  		Fig. 2. Induction of Fas expression in HT29 human colon carcinoma cells after 2 h exposure to 1, 3, 5, or 10 units/ml IFN-{gamma}. Data were derived by FACS analysis and represent the means of duplicate determinations.

 
Pharmacokinetic Analysis of IFN-{gamma}.
On day 1 venous blood samples for pharmacokinetic analysis were drawn after the first IFN-{gamma} dose. Blood samples (5–7 ml) were collected in K3EDTA tubes at pretreatment and at 1, 2, 4, 6, 8, 24, and 48 h after the first IFN-{gamma} dose. Blood was centrifuged at 800 x g for 10 min, and plasma was decanted and stored at -20° until analysis. IFN-{gamma} concentrations were measured by ELISA (R&D Systems, Minneapolis, MN). Thawed plasma (200 µl) was added to a microplate precoated with a polyclonal antibody to IFN-{gamma}, incubated according to vendor instructions, and IFN-{gamma} plasma concentrations were determined based on a standard curve generated by measuring absorbance at 450 nm. A one-compartment model with first order absorption and an absorption delay was fit to the IFN-{gamma} concentration time data using maximum a posteriori as implemented in ADAPT II (23) . Model parameters estimated included volume of distribution (Vc), elimination rate constant (ke), absorption rate constant (ka), and absorption delay (Tlag). Secondary parameters included (AUC0->{infty}), observed maximum concentration (Cmax), and observed time of maximum concentration (Tmax).

Pharmacokinetic Analysis of FUra.
For FUra, serial plasma samples were obtained before FUra administration and at 5 min, 15 min, 30 min, 1 h, 1.5 h, and 2 h after FUra on days 1 and 5. Five to 7 ml of venous blood were drawn into K3EDTA tubes and centrifuged at 800 x g for 10 min. Plasma was decanted and stored at -20° until analysis. The samples were deproteinized with ethyl acetate:isopropyl alcohol, vortexed, and centrifuged for 10 min. The supernatant was evaporated to dryness under nitrogen, reconstituted in mobile phase, and analyzed by reversed-phase high-performance liquid chromatography (Waters Bondapak C18 analytical column) with UV detection using an isocratic mobile phase [2.5 mM ammonium acetate, 2.5% methanol (pH 6.0)] according to published procedures (10) . A one-compartment model was fit to the FUra plasma concentration versus time data (23) . Model parameters estimated included the volume of the central compartment (Vc) and the elimination rate constant (ke). Using standard equations both systemic clearance (C1) and area under the plasma-concentration curve (AUC0->{infty}) were calculated from the parameter estimates. A paired t test was used to assess differences in FUra Cl on days 1 and 5.

Analysis of Fas Expression in Peripheral Blood Samples.
Venous blood (3 ml) was collected into K3EDTA tubes before IFN-{gamma} administration and at 2, 8, 16, 24, 48, and 96 h after the first IFN-{gamma} dose; 100 µl aliquots were stained according to vendor recommendations and immediately analyzed by FACS (Becton Dickinson FacsScan); 5000 cells were collected from the lymphocyte fraction and analyzed with CellQuest software. Anti-CD45, a general leukocyte marker, was used to gate out remaining RBCs and debris, and to monitor the quality of staining. Isotype antibodies with the appropriate fluorescent dyes were used as negative controls. To separately analyze the relative level of cell surface Fas in different types of leukocytes the following directly labeled mAb (Becton Dickinson) combinations in triple staining were applied using anti-CD45-PerCP, anti-Fas-PE, and mAbs (-FITC) to detect specific cell types. Throughout the study the same lot (M044107) of PE-labeled anti-Fas (CD95) mAb (clone DX2; PharMingen #33455X) was used. The individual cell types were analyzed as follows: T cell (anti-CD3-FITC), T helper (anti-CD4-FITC), T cytotoxic (anti-CD8-FITC), B cell (anti-CD19-FITC), natural killer cell [anti-CD56-FITC (IgG2b)], monocyte [anti-CD14-FITC (IgG2b)], and granulocyte [anti-CD15-FITC (IgM)]. At each measurement the PE staining was calibrated with Quantibright (BD) beads. Mean fluorescence intensity was converted to number of PE molecules using Quantiquest software, and relative changes in the cell surface PE numbers were determined.

Clonogenic Assays.
HT29 cells, originally obtained from American Type Culture Collection, were plated at a density of 1500 cells/well in six-well plates in dTHd-free, folate-free, RPMI 1640 containing 10% dialyzed fetal bovine serum (dTHd-free) and 80 nM [6RS]5-methyltetrahydrofolate, as described previously (9) . After overnight attachment, cells were treated with concentrations of FUra of 3 µM, 10 µM, 19.2 µM, or 38.5 µM in the presence of LV (1 µM), and either in the absence or presence of IFN-{gamma} (1–30 units/ml), for 24 h, 8 h, 4 h, and 2 h, respectively. Clonogenic survival was determined at 6 days (the equivalent of seven doublings) after removal of drugs, as described previously (9) .

Statistical Methods.
Fas levels, as measured by the mean fluorescence intensity converted to number of PE molecules, were obtained at intervals after administration of the first and second doses of IFN-{gamma}; levels of interest were those taken at pretreatment (0 h), 8, 24, 48, and 96 h. Fas up-regulation at each time point was measured by the ratio of the Fas level obtained at a given time point to that observed at 0 h.

Fisher’s exact test was used to examine the association between best response and dose of IFN-{gamma}. The exact Wilcoxon rank sum test was used to investigate the association between basic Fas expression level (and also Fas up-regulation, AUC, and duration of exposure >1 unit/ml) with categorical variables including gender, race, response, and presence of grade 3 and 4 toxicity. The Spearman correlation coefficient was used to examine the relationship between Fas expression and up-regulation with continuous variables such as pharmacokinetic parameters and age at diagnosis. The Kruskal-Wallis test was used to explore differences in Fas up-regulation and pharmacokinetic parameters with dose. SAS and StatXact were used for statistical analyses. Results were exploratory, and no adjustments were made for multiple comparisons. For normally distributed data, summary statistics are presented as means and SDs; otherwise, medians and ranges are given.


   Results
Top
 ABSTRACT
 Introduction
 Patients and Methods
 Results
Discussion
 REFERENCES
 
IFN-{gamma} Schedule.
We elected to use a standard regimen for treating patients with FUra and LV. We based our selection of the IFN-{gamma} scheduling on the results of our preclinical studies that demonstrated a sustained rise and maintenance of cell surface Fas expression after a relatively short exposure of HT29 human colon carcinoma cells to the cytokine. Fig. 2Citation demonstrates that after 2-h exposure to 1, 3, 5, or 10 units/ml IFN-{gamma}, Fas was elevated by 1.5-fold at 1 unit/ml, by 2-fold at 3 units/ml, and by 3-fold at 5 and 10 units/ml. Elevation of Fas was maximal at 24 h and was followed by a gradual decline. Based on these results, we chose to administer IFN-{gamma} in patients on days 1, 3, and 5, because a sustained elevation in Fas expression could be anticipated for at least 48 h after a single IFN-{gamma} dose.

Patients and Treatment.
Of 25 patients with metastatic GI cancers enrolled in this trial, 21 had colorectal cancer, 2 had gastric cancer, and 2 had pancreatic cancer (Table 1)Citation . The patients were between the ages of 39 and 81 (median age 65 years) and had good performance status (ECOG 0 or 1). Nineteen patients had prior exposure to FUra. Four colorectal cancer patients were chemo-naive. The median previous treatment regimens before protocol entry was 1 (range, 0–3). Two patients received one cycle of therapy because of early progression of disease in both; 16 patients received two cycles of therapy, 4 patients received four cycles, 1 patient five cycles, 1 patient received six cycles, and 1 patient received seven cycles of therapy. The most frequent reason for treatment discontinuation after two cycles of therapy was PD, except in patients receiving the 100 µg/m2 dose level of IFN-{gamma}, where 3 of 9 patients developed grade 3 toxicity.


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  		Table 1 Patient characteristics

 
Toxicity.
The majority of patients demonstrated manageable grade 2 toxicities typical of FUra/LV containing regimens. Stomatitis and fatigue were most frequently reported (Table 2)Citation . Other GI toxicities including nausea, vomiting, diarrhea, and anorexia were commonly reported. One patient had grade 3 hand-foot syndrome that resolved after supportive measures. The acute toxicity of IFN-{gamma} was minimal and was limited to fever, chills, and myalgias, all of grade 1 on the day of administration. Hematologic toxicity was limited to asymptomatic neutropenia with the nadir leukocyte count typically occurring 14–21 days after initiation of treatment. Two of 7 patients receiving 75 µg/m2 of IFN-{gamma} required dose reduction to 50 µg/m2 on subsequent cycles. One patient received one additional cycle and the other received four additional cycles with an additional dose reduction required. The DLT, stomatitis, was reached at 100 µg/m2 IFN-{gamma}. The temporal pattern of stomatitis for all 3 of the patients was similar with ulceration of oral mucosa beginning day 6 of therapy. Peak stomatitis developed on day 9–10, and healing occurred over the next 5–7 days. All 3 of the patients treated at this dose level on the day 1, 3, and 5 schedule required dose reduction after one cycle of therapy. An additional one to three cycles at 75 µg/m2 were tolerated without development of recurrent grade 3 toxicity, and none of these patients were removed from study for toxicity. Therefore IFN-{gamma} at 75 µg/m2 on days 1, 3, and 5 in conjunction with FUra 375 mg/m2 and LV 200 mg/m2 d x 5 was considered the MTD.


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  		Table 2 Toxicity

 
In an effort to reduce toxicity while preserving the biological effect of IFN-{gamma}, an additional cohort of patients was enrolled with the schedule changed to IFN-{gamma} 100 µg/m2 on days 1 and 3 only. Toxicity for this schedule is shown in Table 2Citation . Stomatitis remained the most common side effect of treatment. Three of 6 patients required dose reduction to 75 µg/m2 and were able to complete an additional one to four cycles of therapy, whereas 1 patient had rapid progression of disease after completion of the first cycle of chemotherapy. Two patients completed two and four cycles IFN-{gamma} at 100 µg/m2 administered on days 1 and 3 without dose reduction. Lack of prior chemotherapy did not influence the ability to increase the tolerated dosage of IFN-{gamma}, as 2 of 4 patients in the additional cohort who were chemo-naive required dose reductions for GI toxicity. On the basis of these results, it was not deemed feasible to continue IFN-{gamma} at 100 µg/m2 for two doses during FUra/LV treatment because of the occurrence of grade 3 toxicity.

Treatment Outcome.
The results of the response to therapy are summarized in Table 3Citation . SD or MR was reached in 2 of 9 patients receiving IFN-{gamma} at a dose level of <=50 µg/m2 and in 4 of 12 patients receiving IFN-{gamma} at >=75 µg/m2. Although statistical significance was not achieved (P = 0.66), greater efficacy was suggested at the higher IFN-{gamma} doses. The median time to progression was 5.5 months for patients with at least SD after two cycles compared with 2 months for those who progressed at the time of first disease evaluation. Of the 6 of 21 previously treated patients demonstrating SD or MR, 5 had colorectal carcinoma and 1 had gastric cancer. Two patients with colorectal carcinoma had received prior adjuvant therapy with FUra/LV only, 1 had received adjuvant FUra/LV and irinotecan first line in the metastatic setting, and 2 had received first line metastatic therapy with FUra/LV followed by irinotecan as second line treatment. The patient with gastric carcinoma had received prior therapy with carboplatin and FUra.


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  		Table 3 Patient response

 
Among the 6 patients receiving 100 µg/m2 of IFN-{gamma} on days 1 and 3 only, 2 previously treated patients received two cycles of therapy and demonstrated PD. The other 4 patients all had colorectal carcinoma and were chemo-naive. One patient with ECOG performance status 2 had rapid progression of disease after one cycle and was referred to hospice care. Two patients achieved a PR, each lasting 4 months, and 1 patient achieved a CR, which lasted for 5 months before recurrence. Two of the 3 responders had dose reduction of IFN-{gamma} to 75 µg/m2 after cycle 1 for stomatitis.

Pharmacokinetics of FUra.
FUra disposition was determined in plasma over the first 2 h after i.v. administration on days 1 and 5 (Fig. 3)Citation , and pharmacokinetic parameters were modeled (Table 4)Citation . The initial plasma concentration of FUra was in excess of 100 µM in all of the patients, and concentrations declined with a half-life of 10–15 min. The disposition of FUra was identical on days 1 and 5. No statistically significant difference in FUra systemic clearance was noted in patients at either the 75 µg/m2 or 100 µg/m2 dose level of IFN-{gamma}. The median clearance on days 1 and 5 for patients receiving 75 µg/m2 IFN-{gamma} was 44 and 37 liter/m2/h, respectively (P = 0.17). The median clearance on days 1 and 5 for patients receiving 100 µg/m2 IFN-{gamma} was 45 and 40 liter/m2/h, respectively (P = 0.5). Concentrations of FUra >10, 25, and 50 µM were achieved for ~1 h, 0. 7 h, and 0.5 h, respectively.



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  		Fig. 3. Mean concentration-time profiles on days 1 ({blacktriangleup}) and 5 (•) of FUra (370 mg/m2 i.v. bolus) in 7 patients receiving 75 µg/m2 IFN-{gamma} s.c. on days 1, 3, and 5. Each data point represents the mean; bars, ±SD.

 

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  		Table 4 Pharmacokinetic parameters for FUraa

 
Pharmacokinetics of IFN-{gamma}.
After s.c. IFN-{gamma} administration at dose levels of 75 µg/m2 or 100 µg/m2, a lag phase of ~1 h in the appearance of the cytokine in plasma was noted. Furthermore, peak concentrations of 2.8 units/ml and 5.7 units/ml, for the 75 µg/m2 and 100 µg/m2 doses, respectively, occurred at 6 h (Table 5)Citation . At a dose level of 75 µg/m2 IFN-{gamma}, plasma concentrations of the cytokine were sustained above 1 unit/ml for 16 h. Patients treated with 100 µg/m2 IFN-{gamma} exhibited plasma concentrations of IFN-{gamma} that remained above 1, 3, and 5 units/ml for 28.5, 13, and 6.5 h, respectively. The median IFN-{gamma} AUCs at 75 µg/m2 and 100 µg/m2 were 41.6 units/ml*h and 95.3 units/ml*h, respectively.


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  		Table 5 Pharmacokinetic parameters for IFN-{gamma}a

 
Statistical Analyses of IFN-{gamma} Pharmacokinetics.
IFN-{gamma} pharmacokinetics (both AUC and duration of exposure >1 unit/ml) were significantly correlated with dose (P = 0.001 and P = 0.003, respectively) and toxicity (presence of grade 3 or 4 toxicity; P = 0.005 and P = 0.008, respectively). Patients who received higher doses of IFN-{gamma} demonstrated higher median AUCs and exposure durations. Similarly, patients with grade 3 and 4 toxicity had higher IFN-{gamma} AUC and exposure durations compared with patients who did not experience toxicity. No associations between IFN-{gamma} pharmacokinetics and best response were observed (P = 0.34 for AUC and P = 0.98 for exposure duration). In this analysis, a patient’s best response overall courses received was used as the outcome of interest. The majority of patients progressed (16 of 25 patients). CR (n = 1), PR (n = 2), MR (n = 3), and SD (n = 3) were grouped together as markers of activity (36%).

Influence of IFN-{gamma} on Fas Expression in Cells within the PBMC Compartment.
In an attempt to determine the biological effect of IFN-{gamma} on elevating Fas expression in cells derived from patients, seven different cell populations within the PBMC compartment were examined by FACS analysis for Fas expression at pretreatment, at various times during the first cycle of treatment, and after the first and second doses of IFN-{gamma} at dose levels from 25 µg/m2 to 100 µg/m2 (Fig. 4)Citation . Dose-related increases in cell surface Fas expression were determined in all of the cell populations within the PBMC compartment, although these were highest in CD15+ and CD19+ cells. The most consistent elevation in Fas expression was demonstrated in CD15+ cells, which comprised a predominant PBMC component (Fig. 5)Citation . Fas expression was elevated by 1.5-fold at 25 µg/m2 and by 2-fold at 50 and 75 µg/m2 IFN-{gamma}, being maximally elevated at 24 h after the first IFN-{gamma} dose, with a sustained elevation for up to 96 h examined. However at 100 µg/m2 IFN-{gamma}, elevated Fas expression (> 1.5-fold) was detected at 8 h after the first IFN-{gamma} dose and continued to rise to a level of 2.5-fold by 96 h. The highest dose of the cytokine gave the most consistent and sustained elevation in Fas expression. From these data, we concluded that the third dose of IFN-{gamma} would not be required on day 5 because Fas expression was already elevated at the time that the last doses of FUra and LV were administered. Hence, an additional 6 patients were enrolled on the trial for examination of the toxicity of cycles of therapy using IFN-{gamma} administration on days 1 and 3 only in each treatment cycle. Furthermore, based on these findings, statistical analyses were limited to investigation of Fas expression and Fas up-regulation in the CD15+ cell compartment.



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  		Fig. 4. Influence of IFN-{gamma} treatment on cell surface Fas expression for up to 96 h after two IFN-{gamma} doses of 100 µg/m2 on days 1 and 3 in a representative patient in CD3+, CD4+, CD8+, CD14+, CD15+, CD19+, and CD56+ cells within the PBMC compartment. Data represent the mean of duplicate determinations. The table shows changes in Fas expression (mean ± SD) in the 7 cellular compartments in all of the patients (n = 6) receiving 100 µg/m2 IFN-{gamma}.

 


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  		Fig. 5. Influence of IFN-{gamma} dose (25–100 µg/m2) on cell surface Fas expression in CD15+ cells. Each data point represents the mean derived from between 3 and 7 patients; bars, ±SD.

 
Statistical Analyses of Fas Expression in CD15+ Cells.
Although mean Fas up-regulation increased by dose, there were no statistically significant differences among dose levels at any time point (0.114 <= P <= 0.37). Patients receiving 100 µg/m2 of IFN-{gamma} had the highest Fas up-regulation values compared with the other dose levels. No Fas data were available from patients receiving 10 µg/m2 of IFN-{gamma}. Fas up-regulation at 8, 24, and 48 h was significantly associated with IFN-{gamma} AUC (P <= 0.036) and also with time of IFN-{gamma} exposure >1 unit/ml (P <= 0.034). All of the associations were positive, meaning that higher values of the pharmacokinetic parameter corresponded with higher measures of Fas up-regulation. Fas up-regulation at 24, 4,8 and 96 h was also significantly associated with toxicity (P <= 0.032). At all times, mean Fas up-regulation was higher for patients with toxicity (Fig. 6)Citation . There was no evidence of associations observed between pretreatment Fas levels with gender (P = 0.96), race (P = 0.19), or age (P = 0.64). Similarly, no evidence of associations between Fas up-regulation and patient demographics was observed (at all times, 0, 8, 24, 48, and 96 h; P >= 0.34). Finally, no correlation was found between elevated Fas expression at 96 h after the first dose of IFN-{gamma} in CD15+ cells and treatment outcome as defined by those patients demonstrating SD, MR, PR or CR (9 patients; P = 0.96).



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  		Fig. 6. Fold increase in Fas expression determined in CD15+ cells at 24 h after the first dose of IFN-{gamma} (25–100 µg/m2) versus response or detection of grade 3 and 4 toxicity.

 
Influence of Pharmacologic Concentrations of FUra and IFN-{gamma} in HT29 Cells.
At an IFN-{gamma} dose level of 75 µg/m2, 1 unit/ml of IFN-{gamma} was maintained in plasma for 16 h, and after 100 µg/m2 IFN-{gamma}, concentrations of the cytokine >1, 3, and 5 units/ml were sustained for 28.5, 13.0, and 6.5 h, respectively. Calculation of IFN-{gamma} concentrations and durations of exposure that would yield an AUC of 41.6 units/ml*h, which was measured in patients after 75 µg/m2 of IFN-{gamma}, ranged from 2 to 23 units/ml for exposure times from 24 h to 2 h. Exposure of HT29 cells in vitro to these IFN-{gamma} concentrations yielded an elevation in Fas expression after 2-h exposure to the cytokine, with maximal elevation at 24 h and up to 3-fold up-regulated expression at concentrations of 5 units/ml and 10 units/ml of IFN-{gamma} (Fig. 2)Citation . A similar profile of elevated Fas expression was determined for the same concentrations of IFN-{gamma} and exposure times of 6.5 h and 24 h as measured in patient samples (data not shown). Hence, Fas expression could be up-regulated in response to IFN-{gamma} at pharmacologically achievable concentrations and durations of IFN-{gamma} exposure in human colon carcinoma cells in vitro, as well as in CD15+ cells derived from patients. The influence of IFN-{gamma} on FUra/LV-induced cytotoxicity was examined in HT29 cells by clonogenic assay for short FUra exposures (2 h) of both IFN-{gamma} (1–30 units/ml) and high FUra concentrations (38.5 µM), and for concentrations of 19.2 µM for 4 h, 10 µM for 8 h, or 3 µM for 24 that would yield a FUra AUC of 76.9 µM*h, which was achieved in the plasmas of patients (Fig. 7)Citation . It was evident that FUra/LV-induced loss in clonogenic survival was potentiated by IFN-{gamma} at clinically relevant concentrations. Short exposures (2 h) to high concentrations of FUra (38.5 µM) combined with IFN-{gamma} were the most effective, yielding >80% loss in clonogenic survival at 5 units/ml IFN-{gamma}, demonstrating that concentrations of the agents achievable in patients and clinically relevant exposure durations were also cytotoxic to human colon carcinoma cells in vitro.



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  		Fig. 7. Clonogenic survival in HT29 cells after (A) 2-h exposure to FUra (38.5 µM); (B) 4-h exposure to FUra (19.2 µM); (C) 8-h exposure to FUra (10 µM); or (D) 24-h exposure to FUra (3 µM) combined with LV (1 µM) and varied concentrations of IFN-{gamma} (1–30 units/ml) simultaneously with FUra. Data represent the mean of three determinations; bars, ±SD.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Patients and Methods
 Results
 Discussion
REFERENCES
 
In this trial we demonstrated that IFN-{gamma} added to a standard regimen of FUra combined with LV was well tolerated in both previously treated and untreated patients, and that the maximum tolerated dose of IFN-{gamma} was established at 75 µg/m2. Regimens containing FUra and LV are considered standard therapy for the treatment of patients with advanced colorectal cancer. The rationale for the Mayo Clinic regimen of combining FUra with LV is based on preclinical studies. The addition of LV to FUra enhances the antithymidylate effect of FUra by elevating intracellular pools of reduced folates, in particular 5,10-methylenetetrahydrofolate, which increases the formation and stability of the ternary complex formed between thymidylate synthase, the active metabolite of FUra, 5-fluoro-dUMP, and 5,10-methylenetetrahydrofolate. The stabilization of the ternary complex thereby increases the level and duration of inhibition of the enzyme and promotes the induction of thymineless death (10) . In patients with advanced colorectal cancer, the FUra/LV combination has demonstrated significant increases in responses rates from 7–15% with FUra alone (with minimal survival benefit) up to 48% when FUra is combined with LV (1, 2, 3, 4, 5) , and in certain studies has enhanced patient survival (1 , 5) . The therapeutic index of FUra is enhanced by LV because FUra toxicity to normal GI tissues appears to be predominantly RNA-mediated (11 , 12) . More recently, newer agents such as the third generation platinum compound, oxaliplatin, or the topoisomerase I inhibitor, irinotecan, have been combined with FUra/LV. In two large randomized Phase III clinical trials comparing the efficacy of FUra/LV combined with oxaliplatin to the efficacy of FUra/LV alone, progression-free survival and overall survival were either modest or no different from those obtained with FUra/LV (24 , 25) . The efficacy of FUra/LV combined with irinotecan versus FUra/LV alone was examined in two prospective, randomized, multicenter Phase III trials in patients with previously untreated metastatic colorectal cancer (6 , 7) . Response rates, time to tumor progression, and survival time for the triple versus the double combination were modestly improved. However, more intensive chemotherapy was delivered in the FUra/LV/irinotecan arm, which resulted in enhanced toxicities (7) , and based on preclinical studies, suboptimal FUra/LV treatment regimens were used including very low doses of LV (20 mg/m2; 6 ).

Rather than combining cytotoxic agents for the treatment of colorectal cancer, we have developed the approach of modulation of the expression of a specific gene, Fas, and hence its signaling pathway, targeted specifically to sensitize colon carcinoma cells to FUra/LV in a Fas-dependent manner, based on rational preclinical design. We demonstrated that FUra/LV cytotoxicity, dependent on DNA- but not RNA-mediated damage, was potentiated by IFN-{gamma} in a Fas-dependent manner because of up-regulation of cell surface expression of the death receptor Fas and enhancement of the Fas signaling pathway, and was independent of the p53 tumor suppressor gene (9) . This is of importance because: (a) the anti-TS effect of FUra is a critical determinant of the sensitivity of colorectal cancers to FUra/LV (1, 2, 3, 4, 5) ; (b) IFN-{gamma} does not sensitize cells to FUra/LV when the mechanism of FUra action is RNA-directed (9) ; (c) Fas dependency of colon carcinoma cells in sensitization to FUra/LV may be unique to this histotype, because other agents that damage DNA including doxorubicin, topotecan, and VP-16 exert their cytotoxic mechanism independent of Fas (26) ; (d) p53 is mutated in >75% of colorectal cancers (21) ; and (e) Fas is reduced in expression in ~50% of colorectal cancers (15 , 16) and is up-regulated after treatment with IFN-{gamma} (9) . These data suggested that we might be able to increase the therapeutic index of FUra/LV in patients for the treatment of colorectal cancer by gene-specific modulation. To this end we initiated a Phase I trial with the goals being to determine the safety and feasibility of combining IFN-{gamma} with FUra/LV, to determine whether the concentrations and durations of exposure to IFN-{gamma} measured in plasma would elicit effects on Fas expression both in patients and in cultured human colon carcinoma cells, and to determine whether FUra/LV cytotoxicity could be potentiated.

The DLT of stomatitis, which occurred at 100 µg/m2 of IFN-{gamma}, necessitated reducing the dose of IFN-{gamma} to 75 µg/m2 in subsequent treatment cycles. Even when the number of doses of IFN-{gamma} was reduced from three to two in each treatment cycle, a dose reduction in the cytokine after the first cycle of therapy was warranted in 3 of 5 patients, 2 of whom were chemo-naive. However 1 patient treated previously and 1 chemo-naive patient received two to four cycles of therapy maintaining the dose of IFN-{gamma} at 100 µg/m2. It is possible that in previously untreated patients the FUra/LV/IFN-{gamma} combination may be better tolerated at the higher IFN-{gamma} dose, but that remains to be determined.

Of particular interest was that in 4 of 12 patients treated previously we observed MRs or SD. These patients were receiving doses of IFN-{gamma} >= 75 µg/m2 in contrast to 2 of 9 patients at lower IFN-{gamma} dose levels, with a minimum of two cycles of therapy. Although these response rates were not statistically significant by dose level, higher activity at the higher IFN-{gamma} dose levels was suggested. Furthermore, 3 of 3 chemo-naive patients treated with two doses of IFN-{gamma} in each treatment cycle demonstrated CR or PR at these higher dose levels of IFN-{gamma}, suggesting that additional evaluation of this regimen in Phase II is warranted. Moreover, despite small sample sizes, significant correlations were demonstrated between the IFN-{gamma} plasma AUC and duration of IFN-{gamma} plasma concentrations: >1 unit/ml versus grade 3 and 4 toxicities. The elevation of Fas expression in CD15+ cells was generally higher at higher IFN-{gamma} doses without reaching statistical significance. However, the actual plasma IFN-{gamma} levels measured by AUC and duration of >1 unit/ml did significantly correlate with the level of Fas up-regulation in this cell population.

Of importance in this study was the observation that the type II IFN, IFN-{gamma}, did not affect the clearance of FUra from plasma. This is in contrast to the observation of Grem et al. (27) that the clearance of FUra was higher in a cycle containing LV, IFN-{alpha}2a, and IFN-{gamma} than in a prior cycle that contained only FUra/LV/IFN-{alpha}2a and also that in the absence of IFN-{gamma}, IFN-{alpha}2a appeared to decrease the clearance of FUra. However, the patients in the Grem study were treated with doses of IFN-{gamma} that ranged from 100 to 1600 µg/m2, so all but 1 of the patients in whom an increased clearance of FUra was observed when IFN-{gamma} was added to the FUra/LV/IFN-{alpha}2a regimen received dosages of IFN-{gamma} that were higher than we have studied in our patient population. IFN-{alpha}2a has been reported to increase the activity of FUra in colorectal cancer (28) . However, Phase III trials in both the adjuvant and metastatic setting failed to establish a treatment benefit to the triple drug regimen with a significant increase in toxicity (29) . These different types of IFNs bind to different cellular receptors and elicit separate and independent postreceptor signaling mechanisms (30) . In this regard, IFN-{alpha}2a has not demonstrated activity in up-regulating Fas expression in human colon carcinoma cell lines.4 Therefore, IFN-{gamma} appears to function in a very different manner than IFN-{alpha}2a in its ability to enhance the cytotoxic action of FUra/LV.

Peak FUra plasma concentrations >100 µM were obtained, and IFN-{gamma} concentrations between 1 and 5 units/ml were achieved for significant periods of time. IFN-{gamma} concentrations observed after s.c. administration were effective at up-regulating the cell surface expression of Fas in CD15+ cells in vivo and also in HT29 human colon carcinoma cells in vitro. These data suggest that CD15+ cells may serve as an effective surrogate marker for measurement of the biological activity of IFN-{gamma} in patients, in particular for potential grade 3 and 4 toxicities when determined at 24 h after the first IFN-{gamma} dose. However the potential of this parameter to predict patient response was not apparent in this small sample size, and this question must await determination in the Phase II trial. Furthermore, it will be necessary to confirm up-regulation of Fas expression in patient tumor tissues in comparison to CD15+ cells. However, at the concentrations and durations of exposure of FUra and IFN-{gamma} observed in patients, the cytotoxic effects of FUra/LV were potentiated by IFN-{gamma} in HT29 human colon carcinoma cells. It is apparent that exposures to IFN-{gamma} as short as 2 h are sufficient to elevate Fas expression in malignant cells, and significant IFN-{gamma} concentrations in excess of 1 and 5 units/ml were achieved for a minimum of 6.5 h. Furthermore, short durations of exposure to FUra/LV at high concentrations, identical to those achieved in the plasmas of patients, are highly cytotoxic to colon carcinoma cells as demonstrated by an 80% loss in clonogenic survival at 3–5 units/ml of IFN-{gamma} and 38.5 µM FUra after 2 h exposures. Lower concentrations of FUra for 4-h or 8-h exposure times were also cytotoxic in combination with IFN-{gamma}, although the effect was decreased when the duration of exposure was increased to 24 h, thereby demonstrating a direct correlation between high FUra concentrations, short exposures, and cytotoxicity.

In summary, this Phase I study was developed from a clear preclinical rationale of the potential for IFN-{gamma} to enhance the therapeutic index of FUra combined with LV for the treatment of advanced colorectal cancer based on the modulation of the expression of a specific gene and a specific death receptor signaling pathway. The treatment regimen was well tolerated, and responses in patients were observed. The results of this trial suggest the strong potential of this treatment regimen to increase response rates and therapeutic outcome in previously untreated patients, and, hence, warrants additional evaluation in Phase II in patients presenting with this disease.


   ACKNOWLEDGMENTS
 
We thank Suzan Hanna for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Awards RO1 CA 32613, RO1 CA 14799, Cancer Center Support (CORE) Grant CA 21765, PO1 CA 23099, the American Lebanese Syrian Associated Charities, and by the Wings Cancer Foundation. Back

2 To whom requests for reprints should be addressed, at Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN, 38105. Phone: (901) 495-3456; Fax: (901) 495-3966; E-mail: janet.houghton{at}stjude.org Back

3 The abbreviations used are: FUra, 5-fluorouracil; LV, leucovorin; GI, gastrointestinal; MTD, maximal tolerated dose; DLT, dose-limiting toxicity; PE, phycoerythrin; FasL, Fas ligand; dTHd, thymidine; AUC, area under the curve; FACS, fluorescence-activated cell sorter; ECOG, Eastern Cooperative Oncology Group; CR, complete response; PR, partial response; MR, minor response; PD, progressive disease; mAb, monoclonal antibody; SD, stable disease; PBMC, peripheral blood mononuclear cell. Back

4 D. M. Tillman and J. A. Houghton, unpublished observations. Back

Received 3/18/02; revised 5/ 1/02; accepted 5/ 3/02.


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 Introduction
 Patients and Methods
 Results
 Discussion
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