Faculty Bibliography

PURPOSE: We tested the hypothesis that polymerase chain reaction (PCR) quantitation of the enzyme thymidylate synthase (TS) within a primary adenocarcinoma of the stomach, has an inverse relationship to response and survival for patients who receive fluorouracil (5FU)-based chemotherapy. PATIENTS AND METHODS: Before systemic chemotherapy, the genetic expression of TS (TSmRNA level) was determined using a PCR method. Gene expression was calculated by determining the ratio between the amount of radiolabeled PCR product with the linear amplification range of the TS gene and the beta-actin gene. Chemotherapy consisted of two cycles of protracted infusion (PI) 5FU 200 mg/m2/d administered for 3 weeks with leucovorin 20 mg/m2/w. Cisplatin 100 mg/m2 was administered on day 1. RESULTS: Sixty-five patients with primary gastric cancer had a median TS mRNA level of 4.6 x 10(-3) (range, 0.9 to 20.1 x 10(-3)). Thirty-five percent of patients had measurable responses in their primary tumors. The mean gastric cancer TSmRNA level in responding and resistant patients is statistically significant (P < .001). The median survival time was 43+ months for treated patients with TSmRNA levels less than the median and 6 months for those with TS m-RNA levels greater than the median (P = .003). CONCLUSION: The genetic expression of TS (TSmRNA level) influences response to 5FU-based chemotherapy and survival for a cohort of patients with primary gastric cancer. Confirmation of these data could lead to therapeutic decisions based on specific molecular properties within a tumor.

The relatively high response rate demonstrated with the use of continuous infusion 5-Fluorouracil (CIFU) 200 to 300 mg/m2 per day in disseminated colon cancer is the rationale behind a NCI-sponsored intergroup trial (INT 0153) for postoperative adjuvant therapy of stage III colon cancer. Because CIFU necessitates a significant reduction in the dose of the modulator leucovorin compared with bolus 5-FU, a pilot study of continuous infusion 5-FU in several doses with levamisole was conducted to determine any unexpected toxicity of this combination, and to assess completion rates of levamisole was conducted to determine any unexpected toxicity of this combination, and to assess completion rates of this dose intensive schedule. CIFU, scheduled as two 12-week cycles, was administered at 250 mg/m2 per day. Pending toxicity at the initial dose, CIFU was to be escalated (300 mg/m2) or de-escalated (200 mg/m2). All but one patient entered on this trial completed 24 weeks of adjuvant CIFU+levamisole. Eight patients (57%) completed 24 weeks of therapy with the 2-week scheduled break. One of these 8 patients required a dose reduction without interrupting the schedule. Six patients had toxicity from the CIFU, which obliged us to interrupt the schedule. Limiting toxicities were stomatitis and hand-foot syndrome. No dose-limiting hematologic toxicity was encountered. Three patients (21%) had catheter problems that required replacement. These data suggest that up to 30% of patients started on this regimen may require dose reduction, shorter infusion courses with rest breaks, or both to complete 24 weeks of adjuvant treatment in order to achieve desired dose intensity.

UFT is an oral preparation combining the 5-fluorouracil (FU) prodrug tegafur (FT) and uracil (U) in a 1:4 ratio, which is commercially available in Japan for the treatment of breast and gastrointestinal cancers. We sought to determine the tolerance of daily oral UFT and to relate this tolerance to the pharmacokinetics of FT and/or the derived FU, while exploring the possibility of circadian FU kinetics contributing to the results. A 28-day schedule followed by 2 weeks rest was began at the initial level of 300 mg/m2/day administered either at 8 a.m. or at 6 p.m. At the following level, 400 mg/m2/day patients were randomly assigned to a split-dose administration or to the above single, timed dose administration. Intolerance to single dosing was clearly demonstrated, and only the split dosing was advanced to 500 mg/m2/day. When this level proved too toxic, 400 mg/m2 was studied further on a 7 a.m., 3 p.m., and 11 p.m. (every 8 h) schedule. Pharmacology was determined on selected patients. In the single dose administration, areas under the curves of FU were higher following p.m. dosing, although substantial interpatient variation was present. Toxicities (diarrhea and neutropenia) were more severe in patients receiving the drug in single daily doses. We conclude that the kinetics of FT are saturable, with disproportionate increases in area under the curve (and toxicities) as dose levels are increased. With divided dosing, tolerance improves. UFT at a dose of 400 mg/m2/day administered as three divided doses (every 8 h) is suitable for Phase II studies, although toxicity requiring cessation of drug administration prior to completion of 28-day cycles will occur in some patients.

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). We have correlated TS protein and gene expression with the response in patients with colorectal (n = 9) and gastric cancer (n = 12) treated with infusional 5-FU plus leucovorin (LV) or infusional 5-FU/LV and cisplatin, respectively. TS protein expression was analyzed by Western blot using TS106 monoclonal antibody and densitometry scanning. TS gene expression was measured by PCR analysis using beta-actin as an internal standard and expressed as a TS:beta-actin mRNA ratio. A close linear relationship was noted between TS protein expression and TS gene expression (r2 = 0.60) for the 21 tumor samples analyzed. TS immunohistochemical staining on 15 of the 21 samples revealed that the TS staining intensity correlated closely with TS protein and mRNA expression. In two biopsy samples, TS protein levels and TS gene expression did not correlate; however, one of these exhibited a focal TS staining pattern. Both the TS protein level and TS gene expression were significantly associated with response to 5-FU-based therapy. Patients with responsive disease had a mean TS protein level of 0.17 +/- 0.03 arbitrary units (range, 0.05 to 0.38), whereas in patients whose tumors did not respond, the mean TS protein level was significantly higher 0.60 +/- 0.09 (range, 0.06 to 1.01; P < 0.01). A similar pattern was noted with TS gene expression. In patients with responsive disease, the mean TS:beta-actin gene ratio was 1.36 +/- 0.3 (range, 0.5-3.3 x 10(-3). In contrast, biopsies from patients with unresponsive disease had a mean TS:beta-actin gene ratio of 15.4 +/- 2.6 x 10(-3) (range, 2.7-35.9; P < 0.01). TS protein and TS mRNA expression are highly correlated, and each predict for response to 5-FU/LV-based chemotherapy in patients with colorectal and gastric cancer.

A clinical trial for patients with measurable, disseminated colorectal cancer is being conducted to determine: (1) if intratumoral expression of thymidylate synthase (TS) affects response to protracted-infusion 5-fluorouracil (5FU); and (2) whether intratumoral expression of TS increases when clinical resistance is found after response to 5-FU. Polymerase chain reaction technology is employed to determine TS expression. Using beta-actin as an internal standard, TS expressions for 26 patients range from 0.5 x 10(-3) to 22.6 x 10(-3). Currently, 22 patients are evaluable for response and TS quantitation of their measurable tumour. 8 patients (36%) have had partial responses; 3 responding patients had been previously treated with 5-FU. A strong statistical association between TS expression and resistance to therapy has been found (P = 0.004). No patient with TS expression of 4.0 x 10(-3) or greater has responded. On average, patients previously treated with 5-FU have slightly higher levels of TS expression in their measurable tumours (P = 0.4). Whether responding patients will develop increased expressions of TS upon clinical progression of their cancer remains to be determined. Confirmation of these results in a larger cohort could lead to a scientific rationale for deciding upon specific therapy for patients with disseminated colorectal cancers.

BACKGROUND: UFT is a fixed-ratio combination of uracil and Ftorafur, a prodrug that is absorbed orally and metabolized in vivo to 5-fluorouracil (5-FU). Uracil potentiates 5-FU through interference with its catabolism. The combination of UFT and leucovorin in patients with advanced incurable colorectal cancer, to evaluate preliminary activity and toxicity in this patient population. METHODS: Twenty-one patients were treated. Twenty patients were evaluable for toxicity and response. Patients received UFT 350 mg/m2/day divided every 8 hours. Patients took a 5 mg tablet of leucovorin every 8 hours, concurrent with each UFT dose. Treatment was continued for 28 consecutive days, followed by a 7-day rest. RESULTS: Five major objective responses (one complete and four partial) were observed. Toxicity was mild, with no dose-limiting myelosuppression. Four patients experienced grade 3 diarrhea or higher, and two patients experienced dose-limiting mucositis. CONCLUSION: UFT and low dose leucovorin is a well tolerated, orally administered regimen with activity in colorectal cancer. A randomized comparison of this regimen with conventional parenteral regimens is warranted.

Intraperitoneal (IP) administration of fluorinated pyrimidines has been evaluated for ovarian and gastrointestinal malignancies in phase I, II, and III trials. The tolerance and pharmacokinetic profile of IP 5-fluoro-2'-deoxyuridine(FUDR) alone and with (R,S)-leucovorin ((R,S)-LV) have each been evaluated in previous phase I studies. FUDR doses of 3 g per day with and without (R,S)-LV doses up to 640 mg per day given IP are well tolerated. The current phase I study was designed to determine the pharmacokinetic profiles and clinical tolerance of escalating doses of the pure biologically active S-isomer of leucovorin ((S)-LV) given IP with the same dosing schedule of FUDR. A group of 16 patients with disease confined to the abdominal cavity were treated in this study. Pharmacokinetic studies of blood and peritoneal fluid, toxicity profiles, and clinical response for the first three cycles are reported here. The toxicity profile did not significantly differ from the prior two studies. All non-hematologic toxicities, such as fatigue, nausea, vomiting, diarrhea, and abdominal discomfort were less than grade 4, and most were less than grade 3. Neutropenia and thrombocytopenia were uncommon and observed only in patients with compromised bone marrow reserve. The pharmacokinetic profiles were also congruent with the previous studies and indicate a three-log advantage for FUDR. The (S)-LV profiles in the peritoneal cavity paralleled those of FUDR. Antitumor effects or absence of progression until after cessation of therapy were documented in 11 patients. At a median follow-up of 18 months 44% of patients were alive. IP administration of 3-g of FUDR and up to 640 mg (S)-LV daily for three days was well tolerated. The tolerance and antitumor effects observed during IP FUDR and LV in these studies encourage further exploration of this regimen against ovarian and gastrointestinal malignancies. The actual role and optimal dose of LV as an enhancer of the antitumor actions of FUDR administered by this route remain unknown.

In a preceding study, we established the tolerance and pharmacokinetic behavior of 5-fluoro-2'-deoxyuridine (FdUrd) given by the intraperitoneal (IP) route. A dose of 3 g daily x 3 days was found satisfactory for Phase II study and exploration of biochemical modulation. Therefore, the current study was conducted to study the tolerance and pharmacokinetics of such a dose-schedule and route of FdUrd combined with escalating doses of leucovorin (LV). Fourteen patients were entered and 13 were evaluable for tolerance determination. Pharmacologic determinations of IP FdUrd and 5-Fluorouracil (FUra) derived from it and LV were obtained by HPLC methods on 11 occasions. Findings were compared with the preceding study of FdUrd alone. LV did not appear to alter the tolerance of IP FdUrd even in the four patients receiving the highest dose of LV (640 mg). Toxicities included nausea, vomiting, and rarely neutropenia and diarrhea. Pharmacokinetic parameters indicate a parallel rate of egress of FdUrd and LV from the peritoneal cavity. The pharmacologic advantage for FdUrd is at least 3 logs as previously reported and one log for LV. Evidence of antitumor effect was noted particularly among untreated patients with gastrointestinal primaries. We conclude that IP FdUrd 3 g and LV in doses of up to 640 mg x 3 days are well tolerated. Since FdUrd is more potent, has an even greater hepatic clearance and shows greater potential for modulation with LV than FUra, it may be the preferred fluoropyrimidine for subsequent studies via the IP route in the treatment of carcinomas with prominent peritoneal spread. The pharmacologic advantage for leucovorin is limited but it is a good marker for peritoneal clearance since it parallels FdUrd clearance.

BACKGROUND: Biochemical modulation of bolus fluorouracil (5-FU) by addition of leucovorin to the treatment regimen has increased response in patients with disseminated colorectal cancer from fewer than 20% to more than 40%. In view of the short half-life of 5-FU and its cell cycle specificity, it may be that infusion rather than intravenous bolus injection would increase efficacy. Furthermore, the advent of safer indwelling intravenous catheters and pump technology, allowing home and ambulatory treatment, has made protracted infusion clinically feasible. To examine these questions, we conducted a phase I trial using protracted infusion of 5-FU by indwelling catheter and pump, with leucovorin given by bolus injection, and reported 40% partial response. PURPOSE: We have now initiated a phase II study of 5-FU given by prolonged continuous infusion with weekly bolus injections of leucovorin in previously untreated patients with measurable, disseminated colorectal cancer. METHODS: Forty-one patients were treated. The regimen consisted of treatment for 4 weeks with 5-FU at a dose of 200 mg/m2 daily as a continuous infusion by indwelling intravenous catheter and pump, followed by a 2-week rest and then by monthly cycles of 3 weeks of treatment and 1-week rest until disease progression. Leucovorin was given as a bolus injection of 20 mg/m2 at the beginning of each week of treatment with 5-FU. RESULTS: Nineteen (46%) of 41 patients had objective response: Three complete responses and 16 partial responses were seen. Overall, the median duration of response was 8 months. The median duration of survival was 16 months: 18 months for responders and 10 months for nonresponders. In general, toxic effects were mild and consisted primarily of stomatitis and palmar-plantar erythrodysesthesia (hand-foot syndrome). Neither grade 4 toxic effects nor treatment-related deaths were observed. The only serious side effects were catheter thrombosis (three patients) and catheter sepsis (one patient). CONCLUSION: We conclude that this safe regimen is one of the most effective for the treatment of disseminated colorectal cancer. IMPLICATIONS: The regimen should be tested prospectively against other regimens in use for this disease. It is currently included in a phase III study of the Southwest Oncology Group for this purpose. That study will assess quality of life as well as response rates and survival duration.