Expression of genes related to activity of oxaliplatin and 5-fluorouracil in endoscopic biopsies of primary esophageal cancer in patients receiving oxaliplatin, 5-flourouracil and radiation: characterization and exploratory analysis with survival.

With a goal of identifying relations between gene expression and response (mucosal or pathological) or survival in esophageal cancer patients (stages II to IV) receiving oxaliplatin, 5-fluorouracil (5FU) and radiation, we measured in endoscopic primary tumor biopsies from 38 patients, the expression of seven genes (gammaGCS, gammaGT, MRP-2, ERCC-1, XPA, TS and DPD) prior to treatment, 1 week following oxaliplatin alone and at the end of the combined radio-chemotherapy cycle using real time QRT-PCR. A higher pretreatment level of XPA was related to shorter survival with a hazard ratio of 2.43 (90% confidence interval 1.09 to 5.43) using Cox regression modeling. However, multivariate analysis with a Cox model indicated low expression of XPA or TS and combined stages II and III had a higher probability of survival (for XPA: hazard ratio 3.0 and 90% C.I. of 1.3 to 6.9, with adjustment for stage included; for TS: hazard ratio is 1.98 with 90% C.I. of 0.94 to 4.20. The expression of TS, gammaGCS, ERCC-1 and MRP-2 declined from D 1 to the end of the cycle (p<0.05, sign test). A validation and further understanding of the findings need to be carried out in a larger study with a more homogeneous population of patients.

Prolonged continuous infusion of fluorouracil with weekly bolus leucovorin: a phase II study in patients with disseminated colorectal cancer.

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.

Phase I study of (6R)-5,10-dideazatetrahydrofolate: a folate antimetabolite inhibitory to de novo purine synthesis.

BACKGROUND: Cancer chemotherapy with folate antimetabolites has been traditionally targeted at the enzyme dihydrofolate reductase and is based on the requirement of dividing tumor cells for a supply of thymidylate and purines. However, a new compound, 5,10-dideazatetrahydrofolate (DDATHF, whose 6R diastereomer is also known as Lometrexol), has become available that prevents tumor cell growth by inhibiting the first of the folate-dependent enzymes involved in de novo purine synthesis, glycinamide ribonucleotide formyltransferase. PURPOSE: We investigated the toxicity and therapeutic activity of DDATHF in a phase I clinical trial. METHODS: DDATHF was given at one of the following dose levels to 33 patients (16 females and 17 males) with malignant solid tumors: 3.0 mg/m2 per week (level A) to 10 patients, 4.5 mg/m2 per week (level B) to 13 patients, or 6.0 mg/m2 per week (level C) to 10 patients. Each drug cycle consisted of three weekly injections of DDATHF followed by a 2-week rest prior to redosing in the next cycle. RESULTS: Of 33 patients, 27 received at least one full cycle of DDATHF. Thrombocytopenia was the major dose-limiting toxicity, and it was severe in one of 10 patients during the first cycle and in two of four patients during the second cycle. Because of cumulative toxicity at 6.0 mg/m2, second or later cycles were abbreviated to two weekly doses. Stomatitis was generally mild, but it was dose-limiting in one patient. Neutropenia was infrequent and mild, and normocytic anemia requiring blood transfusion was common with repeat dosing. Leucovorin was given for grade 2 or greater thrombocytopenia and resulted in hematologic recovery within 1 week in all eight patients so treated. Without leucovorin, the thrombocytopenia lasted from 7 to 49 days in three patients. A partial response was noted in one patient with non-small-cell lung cancer and a minor response in one patient with breast cancer. Three patients with colorectal cancer achieved stable disease for greater than 3 months with improvement in carcinoembryonic antigen levels in one patient. CONCLUSIONS: DDATHF has an unusual pattern of toxicity with repetitive dosing, and humans with advanced cancer are considerably more sensitive than would be predicted from previous animal studies. Although doses of 6.0 mg/m2 per week on our schedule have been determined to be safe, repeated cycles require careful monitoring because of cumulative toxic effects. IMPLICATIONS: Additional phase I studies of DDATHF that relate toxicity to folate intake and tissue folate pools appear warranted.

Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors.

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.

Nonoperative therapy for squamous-cell cancer of the esophagus.

Based on the surgical pathology and survival for patients in previous trials using a neoadjuvant program of chemotherapy (5-fluorouracil [5-FU]-cisplatin) and radiation (3,000 cGy) before surgery for squamous-cell cancer (SCC) of the esophagus, a nonoperative pilot trial was designed to test if survival and recurrence would differ from our historical controls if routine esophagectomy was eliminated. Twenty patients were treated. The protocol called for the delivery of 5-FU infusion (1,000 mg/m2/d X 4 d) days 1 to 4 and 29 to 32 with cisplatin (100 mg/m2) day 1 and 29 sandwiched around external beam radiation (3,000 cGy over 3 weeks). Mitomycin C (10 mg/m2) day 57 was administered with bleomycin infusion (20 U/d X 4 d) days 57 to 60 and 78 to 81. A radiation boost of 2,000 cGy was administered 200 cGy/d days 99 to 103 and 106 to 110. Clinical pulmonary toxicity forced withdrawal of bleomycin and mitomycin C in the last four patients treated; two further courses of 5-FU-cisplatin were administered instead. The median measurement of the 20 esophageal lesions by barium swallow was 7 cm. Four patients underwent salvage surgery to prevent life-threatening aspiration pneumonia. The median survival for the 20 patients is 22 months, with a range from 6 to 39+ months. The six patients clinically without cancer are alive 22+ to 39+ months (median, 35+ months). Three patients died manifesting only local (infield) recurrence; five died manifesting only distant recurrence; and five developed local and distant recurrence. While the toxicity of the four drug regimen as administered was prohibitive, the survival and quality of survival is superior to the regimen previously used, which routinely used surgery after preoperative chemotherapy and radiation.

Preoperative systemic chemotherapy followed by adjuvant postoperative intraperitoneal therapy for gastric cancer: a University of Southern California pilot program.

PURPOSE: A clinical trial for patients with gastric cancer amenable to curative resection was undertaken to determine feasibility and response to preoperative systemic chemotherapy followed by postoperative intraperitoneal (IP) chemotherapy. METHODS AND MATERIALS: Thirty-eight patients with resectable gastric tumor received two cycles of protracted intravenous (IV)-infusion fluorouracil (5FU), 200 mg/m2/d, for 3 weeks with weekly IV leucovorin 20 mg/m2 and IV cisplatin 100 mg/m2 days 1 and 29. Resection of the gastric tumor followed within 3 weeks of completion of systemic chemotherapy. Those who had all visible tumor removed with clear margins received two cycles of IP floxuridine 3,000 mg (total dose) per day for 3 days and IP cisplatin 200 mg/m2 with IV sodium thiosulfate on the fourth day of IP therapy. RESULTS: Thirty-seven of 38 patients (97%) received two cycles of systemic chemotherapy. Thirty-five of 38 patients (92%) underwent laparotomy for gastric tumor resection. Thirty-three patients (87%) had gastric resections performed; 29 (76%) had all visible tumor removed with microscopically negative margins. No operative mortality was encountered. Twenty-six patients (68%) received IP treatment. IV neoadjuvant treatment was well tolerated and resulted in 68% of the patients reporting improvement in abdominal pain, 45% objective remissions by computed tomography (CT), 38% objective remissions by gastroscopy and biopsy, and 8% had complete surgical pathologic response. Neutropenic sepsis during the IP treatment phase contributed to the only treatment-related death. Four of 29 completely resected patients (14%) have had tumor recurrence. The median follow-up time of patients remaining alive is now 19 months. The median survival for 38 patients entered onto this protocol has not been reached at 17+ months. CONCLUSION: This novel approach to the treatment of adenocarcinoma of the stomach is feasible. The neoadjuvant systemic therapy results in significant primary tumor regression. The determination of whether systemic or IP components of the program contribute to decreased recurrence or increased survival awaits a prospectively randomized clinical trial.

Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin.

PURPOSE: Response rates to fluorouracil (5-FU)-based therapy remain low. As new, active agents are being tested, information regarding specific intratumoral genetic determinants of chemotherapy sensitivity or resistance can be used to plan therapy rationally. Intratumoral thymidylate synthase (TS) quantitation may be among the most important determinants of sensitivity or resistance to 5-FU. MATERIALS AND METHODS: Forty-six disseminated colorectal cancer patients had measurable tumor biopsies for polymerase chain reaction (PCR)-based determination of TS mRNA pretreatment. Protracted infusion of 5-FU 200 mg/m2/d for 21 days with weekly intravenous leucovorin 20 mg/m2 each cycle was given. After two cycles, responses were evaluated. Response data were correlated with independently determined intratumoral ratios of TS/beta-actin mRNA for each patient. RESULTS: TS/beta-actin ratios were successfully obtained for 42 patients (91%). TS/beta-actin ratios ranged from 0.3 x 10(-3) to 18.2 x 10(-3) (median, 3.5 x 10[-3]). Twelve patients (26%) responded to treatment (median TS/beta-actin ratio, 1.7 x 10[+3]). Thirty-four patients did not respond (median TS/beta-actin ratio, 5.6 x 10[-3]). No patient with a TS mRNA level greater than 4.1 x 10(-3) responded. The median TS/beta-actin ratio (3.5 x 10[-3]) significantly segregated responders from nonresponders (P = .001). Median survival for patients with TS/beta-actin ratios < or = 3.5 x 10(-3) was 13.6 months; for patients with TS/beta-actin ratios greater than 3.5 x 10(-3), it was 8.2 months (P = .02). CONCLUSION: For this cohort, the intratumoral TS/beta-actin ratio had a statistically significant association with response and survival. This relationship for other 5-FU schedules remains unknown. Confirmation of these data in a larger patient population could lead to determination of therapy for disseminated colorectal cancer based on a specific intratumoral molecular parameter.

Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group study.

PURPOSE: A variety of fluorinated pyrimidine-based regimens for the treatment of disseminated colorectal cancer have been presented in the medical literature. The Southwest Oncology Group designed a screening trial of seven regimens of fluorouracil (5-FU) to assess efficacy and toxicity afforded by biochemical modulation or schedule variations. PATIENTS AND METHODS: Six hundred twenty patients were entered into this trial between August 1989 and January 1993. Eligible patients were classified as having recurrent or disseminated disease that was measurable or nonmeasurable. All eligible patients were evaluated for toxicity and survival; patients with measurable disease were evaluated for response according to standard criteria. RESULTS: No regimen achieved a higher response rate than single-agent bolus 5-FU. Eighty-four percent of patients have been monitored until death. The median survival time for the entire cohort is 14 months. Survival hazards ratios showed a positive trend in favor of the unmodulated infusion regimens, while high-grade toxicities occurred more frequently in the 5-FU bolus arms. The major high-grade toxicities were granulocytopenia and diarrhea. CONCLUSION: In this trial, no regimen provided substantial improvement relative to 5-FU bolus or single-agent therapy for either response or survival in the treatment of disseminated colorectal cancer. The single-agent infusion regimens demonstrated the most encouraging results with a favorable toxicity profile and a 2-month longer survival duration than 5-FU bolus therapy.

Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival.

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.

ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy.

PURPOSE: We have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy. PATIENTS AND METHODS: Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene. RESULTS: The median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) to 19.5 x 10(-3)). Of 17 responding patients, 13 (76%) were less than or equal to 5.8 x 10(-3) and four were greater than 5.8 x 10(-3) (P = .003). The median survival for patients with ERCC1 mRNA levels less than or equal to 5.8 x 10(-3) has not been reached, whereas for those greater than 5.8 x 10(-3) it was 5.4 months (P = .034). The median TS mRNA level, 3.7 x 10(-3) (range, 0.9 to 18.9) also segregated responsive versus resistant tumors (P = .024). With both ERCC1 and TS mRNA levels below their medians, 11 of 13 patients (85%) responded; with both ERCC1 and TS mRNA levels above their medians, two of 10 patients (20%) responded (P = .003). CONCLUSION: Considered separately, either ERCC1 or TS mRNA levels in a primary gastric adenocarcinoma has a statistically significant relationship to response. ERCC1 mRNA levels have a statistically significant association with survival; in this cohort TS mRNA levels did not reach statistically significant association with survival as in our previous publication. Whether these molecular parameters are independent of each other as predictors of outcome remains to be determined.

Phase I and pharmacokinetic study of oral UFT, a combination of the 5-fluorouracil prodrug tegafur and uracil.

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.

High basal level gene expression of thymidine phosphorylase (platelet-derived endothelial cell growth factor) in colorectal tumors is associated with nonresponse to 5-fluorouracil.

The gene expression levels of the nucleoside cleavage enzyme/angiogenic factor thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor, were measured by quantitative reverse transcription-PCR in 38 pretreatment biopsies of colorectal tumors from patients who were subsequently treated with 5-fluorouracil (5-FUra) and leucovorin (LV). The range of TP gene expression (relative mRNA levels) in those tumors nonresponsive to 5-FUra was much broader than that of the responding tumors. In contrast to in vitro studies that had shown that an increased intracellular level of TP potentiates the activity of 5-FUra by converting it to the more cytotoxic nucleoside form 5-fluoro-2'deoxyuridine, tumors with the highest basal TP expressions were nonresponders to 5-FUra/LV therapy. The mean TP mRNA level in the nonresponding tumors was 2.6-fold higher than that of the responding patients. We had shown previously that high expression of thymidylate synthase (TS), the target enzyme of 5-FUra, was also a predictor of nonresponse to 5-FUra (L. Leichman et al, J. Clin. Oncol., 15: 3223-3229, 1997). TP and TS expressions were found to be independent variables in these tumors, so that low expression levels of both TS and TP in tumors predicted a very high response rate (11 of 14) to 5-FUra/LV as well as a significantly longer survival, whereas none (0 of 24) of the patients with high expression of either TP or TS were responders.

Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase.

We had previously shown that high gene expressions (mRNA levels) of thymidylate synthase (TS; Leichman et al., J. Clin. Oncol., 15: 3223-3229, 1997) and thymidine phosphorylase (TP; Metzger et al., Clin. Cancer Res., 4: 2371-2376, 1998) in pretreatment tumor biopsies could identify tumors that would be nonresponsive to 5-fluorouracil (5-FU)-based therapy. In this study, we investigated the association between intratumoral gene expression of the pyrimidine catabolism enzyme dihydropyrimidine dehydrogenase (DPD) and the response of colorectal tumors to the same 5-FU-based protocol. DPD expressions were measured by quantitative reverse transcription-PCR in 33 pretreatment biopsies of colorectal tumors from patients who went on to receive treatment with 5-FU and leucovorin (LV). The range of DPD gene expression in those tumors that were nonresponsive to 5-FU was much broader than that of the responding tumors. None of the tumors with basal-level DPD expressions above a DPD:beta-actin ratio of 2.5 x 10(-3) (14 of 33) were responders to 5-FU/LV therapy, whereas those tumors with DPD gene expressions below DPD: beta-actin ratio of 2.5 x 10(-3) had a response rate of 50%. There was no correlation among DPD, TS, and TP expression values in this set of colorectal tumors, which indicated that these gene expressions are independent variables. All of the tumors that responded to 5-FU therapy (11 of 33) had expression values of all three of the genes, TS, TP, and DPD, below their respective nonresponse cutoff values, whereas, in each of the nonresponding tumors, at least one of these gene expressions was high. The patients with low expression of all three of the genes had significantly longer survival than patients with a high value of any one of the gene expressions. The results of this study show that intratumoral gene expression level of DPD is associated with tumor response to 5-FU and that the use of more than one independent determinant of response permits the identification of a high percentage of responding patients.

p53 and thymidylate synthase expression in untreated stage II colon cancer: associations with recurrence, survival, and site.

We initiated a retrospective study to determine whether p53 status and thymidylate synthase (TS) protein expression in primary colon tumors influence recurrence and survival for patients with stage II colon cancer. Tumor specimens from 45 consecutive untreated patients with stage II colon cancer were examined for p53 and TS protein expression using immunohistochemistry. The median follow-up was 5.1 years. Eighteen patients had left-sided tumors, and 27 had right-sided tumors. Fourteen of 45 patients (31%) developed recurrence. p53 overexpression was detected in the tumors of 18 patients (40%); 10 patients (55%) with p53 overexpression recurred; and 4 of 27 (15%) without evidence of p53 overexpression recurred (P = 0.002). High TS expression was detected in the tumors of 16 patients (36%): 8 patients (50%) with high TS expression recurred, and 6 patients (21%) with low TS expression recurred (P = 0.027). Patients with p53 overexpression had a significantly poorer survival than did those patients without p53 overexpression (P < 0.001). High TS expression was associated with poor survival (P = 0.004). p53 overexpression and high TS expression were significantly associated with left-sided tumors (P = 0.003 and P = 0.022). Thirteen of 16 patients (81%) with high TS expression also overexpressed p53, and 24 of 29 patients (81%) with low TS expression did not manifest p53 overexpression (P < 0.001). p53 and TS expression in primary stage II colon cancer are associated and appear to influence recurrence and survival. In this pilot study, left-sided tumors demonstrate significantly more p53 overexpression and significantly higher TS expression than do right-sided tumors, which may explain the significantly poorer survival for patients with left-sided tumors.

p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival.

Recent studies suggest that there may be a strong correlation between the p53 status of a tumor and a patient's response to chemotherapy. Therefore, we determined p53 status in 36 patients with disseminated colorectal cancer by cDNA sequencing and immunohistochemical staining, as well as by the gene expression level of thymidylate synthase (TS), the target enzyme of 5-fluorouracil (5-FU), by reverse transcription-PCR. Ten patients (28%) experienced a clinical response to 5-FU chemotherapy. Overall, TS expression and response to chemotherapy were associated: 9 of 18 (50%) patients with TS < or = 3.0 x 10(-3) responded, compared to 1 of 18 (6%) patients with TS > 3.0 x 10(-3) (P = 0.003). p53 mutations were found in 21 of 36 patients (58%) using cDNA cycle sequencing, and p53 protein overexpression was found in 20 of 32 patients (62%) using immunohistochemistry staining. Overall p53 status and response to chemotherapy were associated: 5 of 10 (50%) patients with wild-type p53 or negative p53 staining experienced a response, but only 5 of 26 (19%) patients with mutant p53 or p53 overexpression responded. TS expression, but not expression of p53, was significantly associated with overall survival (P = 0.002). Patients with wild-type p53 had significantly lower TS levels compared to patients with mutated p53 (P = 0.044). In this study, we also present data linking specific p53 point mutations to TS expression levels and resistance to 5-FU. Although the number of patients is relatively small, these results identify p53 status and TS gene expression as associated with response in disseminated colorectal cancer; independent studies are needed to confirm these findings and to provide information leading to a better understanding of the role of 5-FU-based chemotherapy in the treatment of colorectal cancer.

Phase II study of selumetinib (AZD6244, ARRY-142886) plus irinotecan as second-line therapy in patients with K-RAS mutated colorectal cancer.

BACKGROUND: More than half of colorectal tumors harbor activating mutations in RAS/RAF proteins. Selumetinib (AZD6244, ARRY-142886) is a small molecule kinase inhibitor targeting MEK kinase, downstream of RAS. We examined the efficacy and safety of selumetinib with irinotecan in second-line therapy. METHODS: Patients with K-RAS mutated colorectal cancer, progressing on first-line oxaliplatin-based chemotherapy with bevacizumab, were eligible for this multicenter open-label phase I/II trial. In part A, a dose was determined using a standard "3 + 3" design; in part B, efficacy was determined. The primary endpoint was RECIST response rate. Historical data for irinotecan were used as reference. Secondary endpoints included progression-free survival and overall survival. RESULTS: Thirty-two patients entered the study, and 31 were treated. All had K-RAS exon 2 mutated tumors. In phase I, the recommended oral dose of selumetinib was 75 mg twice per day with intravenous (IV) irinotecan, 180 mg/m² every 2 weeks. Three patients (9.7 %) had partial response . Sixteen patients (51.6 %) had stable disease for ≥4 weeks, including three >1 year. The most common grade 3 adverse events included diarrhea, neutropenia, fatigue, anemia, nausea, and dehydration. The study was terminated before a pre-planned accrual of 45 subjects. CONCLUSIONS: Despite termination before full accrual, the point estimates of RR and median PFS show promising results, suggesting that further investigations of MEK inhibition in the treatment of metastatic colorectal cancer are warranted. Studies combining MEK inhibitors with cytotoxics or other targeted agents may lead to improved clinical activity based on the emerging preclinical data.

Quantitation of intratumoral thymidylate synthase expression predicts for resistance to protracted infusion of 5-fluorouracil and weekly leucovorin in disseminated colorectal cancers: preliminary report from an ongoing trial.

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.

Intraperitoneal 5-fluoro-2'-deoxyuridine (FUDR) and (S)-leucovorin for disease predominantly confined to the peritoneal cavity: a pharmacokinetic and toxicity study.

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.

Biological modification of protracted infusion of 5-fluorouracil with weekly leucovorin. A dose seeking clinical trial for patients with disseminated gastrointestinal cancers.

A clinical trial was designed to find the maximally tolerated dose of weekly leucovorin (LV) that could be combined with 4 weeks of protracted infusion (PI) of 5-fluorouracil (5FU) at a fixed dose of 200 mg/m2. A total of 36 patients with disseminated gastrointestinal malignancies were treated; 9 either progressed or died before receiving 4 weeks of treatment leaving 27 patients evaluable for toxicity and response. 5FU was given as a protracted infusion using an ambulatory infusion pump and indwelling venous access. LV doses included 20, 25, 50, and 75 mg/m2 given as an i.v. push at the time of weekly pump fill with 5FU. In all, 72% of the patients tolerated LV at 20 mg/m2 for 4 continuous weeks, whereas the higher doses required treatment rests prior to 4 weeks. The dose-limiting toxicity at all doses was stomatitis. No significant myelosuppression was seen; diarrhea was infrequent. Overall, 40% of the patients with measurable cancer had partial responses. In view of evidence of biologic and therapeutic effects of these weekly doses of 20 mg/m2 LV with 200 mg/m2 5FU per day given as a protracted infusion over 4 weeks, phase II trials and multimodality studies for patients with gastrointestinal malignancies are being initiated at our institution using this dose and schedule.

Phase I study of AG 331, a novel thymidylate synthase inhibitor, in patients with refractory solid tumors.

PURPOSE: This was a phase I study of AG 331 to determine systemic tolerance and pharmacokinetics following single and multiple escalating intravenous doses. METHODS: The study was an open-label phase I trial that was divided into two components. In phase IA (single dose), six dose levels from 12.5 to 225 mg/m2 were administered to 18 patients (3 at each dose level) and serial blood samples were collected for 72 h. Upon achieving satisfactory pharmacologic parameters, the multiple dosing component (phase IB) was initiated. Six dose levels from 50 to 800 mg/m2 per day were administered for 5 consecutive days to 18 patients. Pre- and postdose blood samples were obtained on days 1-4 and serial blood samples were collected over 24 h following dose 5. Nonhematologic and hepatic toxicities were assessed, serum AG 331 concentrations were measured and pharmacokinetic parameters determined. RESULTS: Other than fatigue, no severe toxicities were encountered in phase IA. Liver toxicity was manifested by elevations in transaminase first noted at multiple doses of 200 mg/m2 per day for 5 days. Fever and malaise but no myelosuppression were noted. The mean terminal t1/2 following single doses was significantly shorter than the t1/2 following multiple dosing (6.8 vs 9.9 h) and clearance was significantly faster following single doses than following multiple dosing (81.7 vs 30.4 l/h), but no significant difference in Vd was noted. CONCLUSIONS: The dose-related toxicity profile precludes further clinical development at this time. The pharmacokinetics of AG 331 following single and multiple doses showed significant differences.