Phase I Study of Capecitabine With Concomitant Radiotherapy for Patients With Locally Advanced Pancreatic Cancer: Expression Analysis of Gen
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《临床肿瘤学》
the Department of Medicine, Division of Hematology-Oncology, Division of Gastroenterology and Hepatology, Division of Radiation Oncology, Department of Pharmacology/Toxicology, and University of Alabama at Birmingham Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Department of Mathematics and Statistics, Auburn University, Auburn, AL
ABSTRACT
PURPOSE: To establish the feasibility of capecitabine with concurrent radiotherapy (XRT) in patients with locally advanced (LA) pancreatic cancer and evaluate the effect of XRT on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-alpha (TNF-).
PATIENTS AND METHODS: Fifteen patients with LA pancreatic cancer received three-dimensional conformal XRT to a dose of 50.4 Gy with capecitabine at escalating doses from 600 to 1,250 mg/m2 bid (Monday through Friday). Following chemo-XRT, stable and responding patients were treated with capecitabine 2,000 mg/m2 orally bid for 14 days every 21 days. Tumor specimens were procured with endoscopic ultrasound–guided fine-needle aspiration 1 week before and 2 weeks after chemo-XRT to evaluate TP, DPD, and TNF- mRNA levels.
RESULTS: Dose-limiting grade 3 diarrhea was observed in two of six patients treated at a capecitabine dose of 1,000 mg/m2 with XRT. Three patients (20%) achieved partial response. Mean percent difference in TP pre- and post-XRT was 119.2% (P = .1934). There was no significant differences in mean TNF-, or DPD levels pre- and post-XRT (P = .1934 and .4922, respectively). TP and TNF- levels were not significantly correlated both at pre- and post-XRT (P = .670 and P < .154, respectively). Median value of TP:DPD ratios at baseline was 2.65 (range, 0.36 to 11.08). No association between TP:DPD ratio and efficacy of capecitabine or severity of toxicities was identified.
CONCLUSION: The recommended dose for phase II evaluation is capecitabine 800 mg/m2 bid (Monday through Friday) with concurrent XRT. This approach offers an easy alternative to intravenous fluorouracil as a radiosensitizer in these patients. Role of TP and TP:DPD ratio warrants further investigation in a larger clinical trial.
INTRODUCTION
Pancreatic cancer, one of the most common gastrointestinal tumors, has a 5-year survival of less than 5%.1 Despite representing only 2% to 3% of the total cancer incidence, it is the fourth leading cause of cancer death in the United States.2,3 The majority of patients have advanced disease at the time of diagnosis, and treatment options are limited for this subset of patients. Standard treatment for locally advanced disease remains fluorouracil (FU; either bolus or continuous infusion) and 50 to 60 Gy radiation (XRT).4,5 The schedule involving continuous infusion of FU requires indwelling catheters and ambulatory pumps and is more difficult to administer on an outpatient basis. Although this combined therapy has been shown to increase local control and median survival from 8 to 12 months,4,5 almost all patients succumb to the disease secondary to recurrence, either local or distant. Another more recent method uses gemcitabine as a radiosensitizer, but the optimal dose of gemcitabine with XRT is still not defined, nor is it known whether this regimen would be superior to FU chemoradiotherapy.6 A new promising chemoradiotherapy approach is to combine capecitabine with XRT.
Capecitabine (Xeloda; Hoffmann-La Roche Inc, Nutley, NJ) is an oral fluoropyrimidine that generates FU preferentially at the tumor site by exploiting the higher activity of the enzyme thymidine phosphorylase (TP) in tumor compared with normal tissue.7 The tumor-selective activation of capecitabine potentially enhances safety by minimizing systemic exposure to FU. In clinical trials, capecitabine monotherapy has demonstrated a favorable safety profile that is typical of infused fluoropyrimidines.7,8 The majority of adverse events were mild to moderate in severity, with a low incidence of alopecia and myelosuppression. As an oral agent, capecitabine can be administered in the outpatient setting while, as a result of rapid and almost complete absorption in the upper gastrointestinal tract, providing a pharmacokinetic profile similar to low-dose continuous infusion of FU.8 This offers a more convenient therapy for patients, avoiding the complications and pain associated with intravenous (IV) therapies. Two different administration schedules were explored in phase I studies9,10: the intermittent schedule (2 weeks of treatment followed by 1 week of rest) with a dosage of 1,250 mg/m2 bid, which was used for further phase III development of capecitabine in colorectal and breast cancer; and a 21-day continuous regimen. The latter study reached a maximum-tolerated dose (MTD) of capecitabine 829 mg/m2 bid, and hand-foot syndrome, nausea, vomiting, vertigo, abdominal pain, diarrhea, and thrombocytopenia were reported as dose-limiting toxicities (DLTs).11
Capecitabine is active in pancreatic cancer as a single agent. Cartwright et al12 performed a phase II trial evaluating capecitabine (2,500 mg/m2/d bid x 14 days, every 3 weeks) in patients with advanced pancreatic cancer. Among 38 assessable patients, there were three partial responses, and 11 patients had stable disease for more than four treatment cycles. Median time to progression was 111 days and median survival was 214 days. Clinical benefit response was positive in 17% of patients and stable in 21% of patients. Responses were as follows: 24% positive responses in pain intensity, 12% in analgesic consumption, and 2% in Karnofsky performance status (KPS). Seven of 42 patients had a decrease of 20 points from baseline in their KPS score, with median time to deterioration of 57 days. Weight was maintained within 10% of baseline. The most common grade 3/4 related adverse events were hand-foot syndrome (17%), diarrhea (17%), and nausea (10%).12
As an oral agent that mimics continuous-infusion FU, capecitabine has the potential to replace IV FU and simplify chemoradiotherapy for pancreatic cancer. Earlier preclinical studies using human colon and breast tumor xenograft models suggested that TP and dihydropyrimidine dehydrogenase (DPD) are the best indicators of tumor response to capecitabine with elevated TP expression resulting in higher intratumor levels of FU.8 Conversely, high DPD levels inversely correlate with intratumor FU because of increased degradation.13 Radiation has been shown to significantly increase the efficacy of capecitabine through induction of TP.14 Of particular interest is that the induction of TP by XRT appears to be tumor associated, and this induction of TP may extend to tumors completely outside the field of XRT (abscopal effects).15 Recent pancreatic xenograft studies from our laboratory demonstrated a synergistic antitumor effect with concomitant capecitabine and XRT in both radiated and contralateral lead-shielded tumors in the same animals.16 Interestingly, these studies also suggest that genes other than TP and DPD may be responsible for the synergistic antitumor effect observed with concurrent administration of capecitabine and XRT.16 Taken collectively, these studies have implications in the rational design of treatment paradigms for pancreatic cancer where metastatic disease remains the primary cause of patient morbidity
We report the results of a phase I dose-escalation study conducted to determine the MTD of oral capecitabine administered concomitantly with standard XRT in patients with locally advanced, unresectable pancreatic cancer. To exploit any potential upregulation of TP secondary to XRT, tumor biopsies before and after XRT were performed.
PATIENTS AND METHODS
The study was conducted according to the principles of the Declaration of Helsinki as amended in Somerset West in 1996, and to good clinical practice guidelines. Approval was gained from the institutional review board, and each patient gave written informed consent before being recruited onto the trial.
Eligibility Criteria
Male or female patients 19 years of age with histologically confirmed pancreatic adenocarcinoma determined unresectable by surgeons (on the basis of vascular involvement as per institution's standard) were eligible for this study. Further inclusion criteria were Eastern Cooperative Oncology Group performance status 0 to 2, adequate bone marrow function defined as absolute neutrophil count 1.5 x 103/mm3, platelet count 100 x 103/mm3 and hemoglobin 9.0 g/dL, adequate renal function defined as a serum creatinine < 1.6 mg/dL, adequate hepatic function defined as a serum bilirubin 2 mg/dL and AST levels 2.5x upper limit of normal, and patients capable of providing written consent.
Exclusion criteria included presence of distant metastases, including liver and lungs, previous chemotherapy, XRT or chemoradiotherapy for pancreatic cancer. The following patients were also excluded: pregnant or lactating patients, women with childbearing potential who lacked a reliable contraceptive method, patients with organ allografts, patients with significant cardiac disease, and patients with CNS metastases or a history of uncontrolled seizures, CNS disorders, or psychologic disability thought to be clinically significant, precluding informed consent or adversely affecting patient compliance. Patients were also excluded if they had serious, uncontrolled infections, malabsorption syndrome, or if they lacked physical integrity of the upper gastrointestinal tract ensuring rapid and reproducible absorption of the drug, and patients with known sensitivity to fluoropyrimidines.
Study Design and Treatment
The primary objective of the study was to determine the MTD of oral capecitabine administered continuously bid in combination with standard XRT in patients with locally advanced pancreatic cancer, using a standard escalation design. Secondary objectives included evaluation of the upregulation of TP and tumor necrosis factor-alpha (TFN-), the safety profile, preliminary assessment of the antitumor activity of the combined-modality treatment, and association of TP:DPD ratio to outcome.
Study treatment was started within 14 days after screening assessment. The study consisted of two parts: part A and part B (Fig 1). During part A, patients received concurrent XRT with escalating doses of capecitabine (Monday through Friday) during a period of approximately 6 weeks until they reached the MTD. After finishing part A, patients were assessed for tumor response and/or resectability to potentially undergo a surgical resection. During part B, stable and responding patients were treated with capecitabine 2,000 mg/m2 orally bid for 14 days every 21 days (one cycle) until evidence of progressive disease, unacceptable toxicity, or patient's preference. Restaging was performed every three cycles (9 weeks). Dose escalation in cohorts of three new patients per dose level using a standard phase I study design. Up to six patients were included in the dose cohort if one DLT was observed in any cohort, or if a maximal dose of 2,500 mg/m2/d was reached. Tumor specimens were obtained by a single experienced endosonographer, with endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) biopsies performed 1 week before and 2 weeks after chemoradiotherapy as previously described.17 Complications were assessed immediately by the endosonographer and at 2 weeks per University of Alabama at Birmingham (UAB) protocol.17
Patients were irradiated once daily, 5 days per week, at 1.8 Gy per fraction, throughout the course of 6 weeks. Computed tomography (CT) image–based three-dimensional treatment planning was utilized to optimize XRT treatment planning by facilitating identification of the target volume and surrounding normal structures (Fig 1). Attempts were made to minimize XRT to surrounding normal tissues while ensuring adequate dose to the target volume. CT simulation was performed with IV and oral contrast material to assist in localizing kidneys, liver, stomach, and intestines. Anatomic structures were contoured for dose-volume histogram (DVH) analysis. The intestines were defined as the contents within the peritoneal cavity, excluding the stomach, spleen, liver, kidneys, aorta, and gross target volume (GTV) to allow for organ motion. The maximum extent of the tumor and involved nodal areas (gross tumor volume), plus adjacent locoregional nodes (ie, celiac, peripancreatic, and portal), and para-aortic nodal areas at risk for residual microscopic diseases (clinical target volume [CTV]) were also defined by CT.
XRT began on the first day of week 1 of the study. The initial target volume received 1.8 Gy/d delivered Monday through Friday for 25 fractions (45 Gy). Typically, the edges of the initial fields were defined superiorly 1.5 cm above the CTV, inferiorly to cover the para-aortic nodes to the L3-4 intervertebral space, laterally and anteriorly with a 1.5-cm margin around the CTV and posteriorly by splitting the anterior vertebral bodies in half. After 45 Gy, an additional three to five 1.8-Gy fractions were delivered to the GTV or tumor bed with a 1.5-cm margin, for a total dose of 50.4 to 54 Gy. XRT was delivered from high-energy linear accelerators with 15-MV photon beams. Patients received 45 to 54 Gy, median dose of 50.4 Gy to the tumor bed.
Dose Modifications
Capecitabine was to be administered at planned escalating doses of 600, 800, 1,000, and 1,250 mg/m2 bid Monday through Friday (weekends off) for the duration of XRT. The first daily dose was administered approximately 2 hours before XRT, with the second dose administered 12 hours after the first. The following recommendations for dose reductions were applied: if a patient experienced a grade 2 or 3 toxicity that was considered possibly related to capecitabine treatment or clearly not related solely to XRT, capecitabine treatment was interrupted and appropriate prophylactic treatment administered. When the toxicity resolved to grade 0 to 1, treatment was resumed without dose adjustment. However, in the case of grade 3 nausea/vomiting or diarrhea not resolving to grade 0 to 1 within 2 days of interruption despite symptomatic treatment, capecitabine treatment was resumed at the preceding dose level without prophylactic treatment. On the recurrence of toxicity at grade 2 or more severe intensity, treatment was interrupted until the toxicity had resolved to grade 0 to 1. Treatment was resumed at the preceding capecitabine dose level (or at the same dose level in patients treated at the lowest dose level). The XRT schedule was not modified unless the severity of the toxicity worsened or a new toxicity of grade 2 or more severe intensity occurred. If a toxicity considered to be clearly related to XRT, such as local skin toxicity, occurred at grade 2 or more severe intensity, XRT was interrupted until the toxicity had resolved to grade 0 to 1 and then resumed with appropriate prophylactic treatment, if required. If the toxicity recurred at grade 2 or more severe intensity, XRT was interrupted until the toxicity had resolved to grade 0 to 1. The administration of capecitabine was not modified unless the severity of the toxicity worsened or a new grade 2 or more severe toxicity developed. If grade 4 toxicities developed, the combination treatment was discontinued, unless the investigator considered it to be in the best interest of the patient to continue treatment with XRT alone or in combination with a reduced dose of capecitabine.
Evaluation of Safety and Efficacy
Adverse events were graded according to National Cancer Institute Common Toxicity Criteria (version 2.0), with the exception of hand-foot syndrome (HFS), which was graded 1 to 3.18 DLT was defined as the occurrence of one or more of the following: any nonhematologic grade 3 toxicity except alopecia, grade 4 vomiting or grade 3 stomatitis, or diarrhea that does not resolve to grade 0 to 2 within 2 days, or grade 3 HFS that does not resolve to grade 0 to 2 within 1 week of starting symptomatic/prophylactic treatment; and grade 4 neutropenia, grade 3/4 neutropenic fever, grade 3 thrombocytopenia, hemorrhage or infection, grade 4 hyperbilirubinemia, or grade 3 shift in liver transaminase concentrations. Adverse events requiring interruption of capecitabine for more than 2 weeks were also classified as dose limiting.
Three patients were recruited to each dose level. The capecitabine dose was escalated when all three patients had completed a minimum of 6 weeks' treatment without DLTs, with at least one patient completing the entire treatment course. If one patient experienced a DLT, an additional three patients were recruited to the same dose level. At dose level three (1,000 mg/m2 bid), six patients were recruited because of grade 3 diarrhea that occurred in one of the first three patients. The MTD was defined as the dose below that caused DLTs in at least two patients in a cohort of six patients.
The safety analysis included all patients who received at least one dose of capecitabine in combination with one dose of XRT. All adverse events were monitored continuously during treatment and for 6 weeks after the end of treatment. Hematology and clinical chemistry was performed weekly during chemo-XRT and thereafter, every 3 weeks during treatment with capecitabine monotherapy. Following chemo-XRT, tumors were evaluated on the basis of Response Evaluation Criteria in Solid Tumors Group 19 criteria at baseline, and 3 to 4 weeks after chemo-XRT.
Tissue Collection, Preparation, and Determination of TP, TNF-, and DPD
Specimens of primary pancreatic ductal adenocarcinoma were obtained via EUS-guided biopsy before starting XRT on day 1 and during week 2 after chemoradiation. Tissues to be utilized for RNA extraction were snap frozen in liquid nitrogen and stored at –80°C. Total RNA was isolated using the Qiagen RNA Purification kit following manufacturer's instruction (Qiagen, Valencia, CA). All sample concentrations were calculated spectrophotometrically at A260 and diluted to a final concentration of 20 ng/μL in RNAse-free water containing 12.5 ng/μL of total yeast RNA (Ambion, Austin, TX) as a carrier.
Real-Time Quantitative Polymerase Chain Reaction
Expression levels were determined using an ABI 7900 Sequence Detection System as previously described by our laboratory.16,20,21 The real-time quantitative polymerase chain reaction (RT-Q-PCR) primers were as follows: human TP forward (5' TCC TGC GGA CGG AAT CC-3'), reverse (5'TGA GAA TGG AGG CTG TGA TGA G-3') and fluorophore-labeled probe (FAM - CAG CCA GAG ATG TGA CAG CCA CCG T-TAMRA); COX-2 forward (5'GAA TCA TTC ACC AGG CAA ATT G-3'), reverse (5'TCT GTA CTG CGG GTG GAA CA-3'), and probe (FAM-TGG CAG GGT TGC TGG TGG TAG GA-TAMRA). The sequence for the primers and probes for human DPD, and S9 ribosomal have been previously described.20,21 Expression levels were calculated using the relative standard curve method.20,21 All reactions were run in triplicate and standard curves with correlation coefficients falling below 0.98 were repeated. Control reactions confirmed that no amplification occurred when yeast total RNA was used as a template or when no-template control reactions were performed.
Statistical Methods
An escalation design with three to six patients was chosen on empiric grounds, according to current standards in phase I cancer trials.22 The chance of not detecting a toxicity that occurs in fact in every second patient is only 1.6% in a cohort of six patients, and less than 0.1% in a cohort of 12 patients. According to the exploratory nature of this pilot trial, only descriptive statistics are presented with respect to response rates, as well as, means with standard deviation (SD), medians and ranges for other quantitative terms. In comparing, pre- and postradiation TP, DPD, and TNF- mRNA levels, the results of a paired t test and signed rank Wilcoxon test are reported. Sample correlation coefficients reported were computed as Pearson's product moment.
RESULTS
A total of 15 patients were accrued onto the study at UAB between April 2003 and March 2004. The demographic characteristics of the patients enrolled are listed in Table 1. The patients had newly diagnosed adenocarcinoma of pancreas, found unresectable on the basis of EUS and CT scan findings as per institutional guidelines. There were no major deviations from the protocol except that one patient (dose level 2) withdrew the consent during therapy, and two others were taken off the study because of reasons unrelated to the study: one patient developed duodenal obstruction and the second patient developed jaundice, work-up of which showed new liver metastases. None of the patients experienced any complications from EUS-FNA after chemoradiotherapy. In particular, none experienced pancreatitis, infection, or hemorrhage.
Dose Escalation and DLTs
Three patients were treated at the lowest dose levels of 600 and 800 mg/m2 bid, respectively, with no DLTs observed. Two of the six patients treated with capecitabine 1,000 mg/m2 bid experienced grade 3 diarrhea, occurring during weeks 4 and 5, respectively (Table 2). Only one patient (1,000 mg/m2 bid) experienced HFS (grade 2) during chemo-XRT. There were no capecitabine dose reductions whatsoever. Apart from the cases mentioned herein and the two patients experiencing DLTs, no capecitabine treatment interruptions were required. Therefore, on the basis of the DLTs, 800 mg/m2 bid Monday through Friday with concomitant XRT is defined as the MTD.
Hematologic Toxicity
No hematologic toxicities except grade 1 anemia were observed during chemo-XRT. All these anemic patients were treated with epoetin alfa without any requirement for blood transfusion. Grade 1 to 2 anemia was also seen during capecitabine alone, not different from that seen in other trials. Leukocytes showed no marked decrease, with the median values staying slightly above the lower limit of the normal range (4.0/nL) throughout the study period. Platelet counts showed a similar decrease, again with no trend toward cumulative toxicity. Counts below the normal range were rare.
Nonhematologic Toxicity
Adverse events according to maximum National Cancer Institute Common Toxicity Criteria are presented for the whole patient group (Table 2). Gastrointestinal toxicities occurred in approximately half of all patients, consisting primarily of grade 1 to 2 nausea (grade 1 in seven patients and grade 2 in three patients) and vomiting (grade 1 in three patients and grade 2 in two patients). Diarrhea occurred in four patients at grade 1, one patient at grade 2, and in two patients at grade 3, the latter being observed at the highest dose level (1,000 mg/m2 bid). According to the study protocol, this event was considered to be a DLT. Grade 1 mucositis was noticed in one patient at 600 mg/m2. Five patients developed grade 1 fatigue and two patients had grade 2 fatigue. Increased values of bilirubin without clinical relevance, common in other capecitabine trials, were recorded in few patients during capecitabine-alone therapy. One grade 2 hyperbilirubinemia at 600 mg/m2 and one grade 1 hyperbilirubinemia at 800 mg/m2 were observed during the chemo-XRT period. Only one of these two patients was found to have metastatic liver disease on evaluation for increased bilirubin. One patient developed grade 2 HFS during week 6, at dose levels of 1,000 mg/m2. No grade 3 HFS was observed during chemo-XRT period. Only one patient developed grade 2 HFS during cycle five of capecitabine monotherapy, which later worsened to grade 3 during cycle eight. Three additional patients had grade 1 HFS. Skin toxicity was observed in approximately 30% of patients (grades 1 and 2), mainly in the irradiated regions and was attributable in most cases to XRT. Also, patients tolerated capecitabine well when administered alone. Except for the observed DLT diarrhea, no clear-cut dose-toxicity relationship was evident.
Antitumor Activity
Among 15 assessable patients, overall response rate was 20%, with three patients achieving a partial response. No patient was found to be resectable because of persistent vascular involvement. Four patients had stable disease after finishing chemo-XRT. Nine patients continued on capecitabine cycles after treatment with concurrent capecitabine and XRT. Median number of cycles administered was six (range, three to 17). Median survival was 14 months, with three patients still surviving at 20 months. Two of these three patients are currently on second-line and third-line chemotherapy, respectively. Most patients received gemcitabine on evidence of progressive disease. Although there was no local disease progression, seven patients developed progressive diseases because of liver metastases.
Quantitation of TP, TNF-, and DPD Expression
TP, TNF-, and DPD mRNA was quantitated in pancreatic tumor tissue by RT-Q-PCR (Tables 3 and 4). The mean percentage of difference in TP expression pre- and post-XRT was 119.2% (median, 61.2%; SD, 175.7%; P = .1934). There was no significant differences in the mean TNF- or DPD levels pre- and post-XRT (P = .1934 and .4922, respectively). TP and TNF- levels were not significantly correlated both at pre- and post-XRT (r = –0.137; P = .670 and r = 0.486; P < .154, respectively). This finding may suggest that mediators other than TNF- may be involved in the upregulation of TP by XRT. The median value of TP:DPD ratios, at baseline, was 2.65 (range, 0.36 to 11.08; Table 5). No association between TP:DPD ratio and efficacy of capecitabine was identified. No correlation was made to overall survival because of the small number of patients. There was no significant correlation between the TP:DPD ratios and the severity of toxicities. None of the patients who developed DLTs were found to be DPD deficient.
DISCUSSION
Two of the six patients treated with capecitabine 1,000 mg/m2 bid experienced grade 3 diarrhea defined as DLT, 1,000 mg/m2 bid Monday through Friday with concomitant XRT was defined as the MTD. Therefore, capecitabine 800 mg/m2 bid Monday through Friday with concomitant XRT is the recommended dosage for subsequent trials.
This is the first human trial in pancreatic cancer that analyzed the intratumor levels of TP and DPD as predictors of tumor response to capecitabine. TP is an enzyme that catalyzes the mutual transformation of the pyrimidine nucleosides thymidine and thymine in nucleic acid metabolism. TP is also an enzyme that converts the FU-based anticancer drugs, 5'-deoxy-5-fluorouridine (5'-DFUR) and its derivative, capecitabine, into FU, and it is, therefore, a limiting factor of the antitumor effects of these anticancer drugs. Capecitabine is a prodrug that is converted to 5'-DFUR in the liver and tumor tissue. Because it is reported that TP is the same protein as platelet-derived endothelial cell growth factor,23 it is considered as a factor involved in tumor angiogenesis and reflects the biologic characteristics of cancer. Prior reports in stomach cancer and colon cancer show that high TP is associated with a poor prognosis,24,25 and that the FU/LV drug regimen is ineffective in colon cancer,26 suggesting that cases of colon cancer with a high TP should be treated with 5'-DFUR and capecitabine. The relationship between the antitumor effects of FU-based anticancer drugs and DPD27 has been noted. Studies on human xenograft models have also shown increased levels of TNF- with increased TP levels14 after XRT. Because TNF- is an upregulator of TP,14 these studies suggest that XRT might increase TP levels indirectly through TNF- in the tumor tissue. Therefore, TP and TNF- expression levels were measured pre- and post-XRT for each patient in our study. The mean difference in TP expression pre- and post-XRT was 119.2 (P = .1934). There was no significant differences in the mean TNF- pre- and post-XRT (P = .1934). And TP and TNF- levels were not significantly correlated both at pre- and post-XRT (r = –.137; P = .670 and r = .486; P < .154, respectively). This finding may suggest that mediators other than TNF- may be involved in the upregulation of TP by XRT.
Recently, preclinical studies on human cancer xenograft models as well as a clinical study using 5'-DFUR in adjuvant chemotherapy in gastric cancer revealed that the efficacy of 5'-DFUR was correlated with the ratio of TP to DPD (TP:DPD) activity.28-30 On the basis of these data, we investigated the relation between TP and DPD expression and the efficacy of capecitabine. No association between TP:DPD ratio and efficacy of capecitabine was identified. There was no significant correlation between the TP:DPD ratios and the severity of toxicities and none of the patients who developed DLTs were DPD deficient.
The results of this phase I study are in accordance with our pancreatic xenograft models demonstrating the synergistic effects of capecitabine with concomitant XRT, but no induction of TP levels in either radiated or contralateral shielded xenografts.16 While these results support the concurrent use of capecitabine and XRT in pancreatic cancer, there appears to be additional genes (other than TP and DPD) associated with response to capecitabine alone and with XRT. The molecular basis of synergy has been reported to be induction of TP expression following XRT.14 Interestingly, our pancreatic xenograft model, which was used to examine response to capecitabine in radiated and contralateral lead–shielded xenografts, demonstrated abscopal effects, with increased antitumor efficacy to capecitabine occurring in tumors outside the field of XRT.16 The precise mechanism(s) by which XRT results in tumor-associated TP induction remains to be elucidated. However, the delay between XRT and the increase in TP expression levels suggests a secondary/mediated induction of TP, rather than a direct effect. Further, the abscopal induction of TP in shielded contralateral xenografts suggests that soluble cell factor(s) may be involved. Initial studies have focused on TNF- and interferon-, which have been shown to both increase after XRT and induce TP expression when added as single agents in vitro.14,31 Taken collectively with analysis demonstrating elevated TP expression in pancreatic tumor compared with normal pancreas, these studies provided the rationale for this phase I clinical study.
There are many methods for the measurement of TP and DPD, including immunohistochemistry, RT-Q-PCR and an enzyme-activity assay.16,20,21,24-26,32 Nishida et al.24 reported that the enzymatic activity of TP closely correlates with the enzyme protein content in the tumor tissues of stomach cancer, colon cancer, and breast cancer (r = .909; P < .001). The immunohistochemical method is the most widely used because of its convenience, but it is difficult to measure the amounts of TP and DPD quantitatively. Instead, we used RT-Q-PCR to measure TP and DPD in this study. This method is also convenient and allows the quantification of enzyme activity in small pieces of tissue such as endoscopic biopsy specimens. These quantitative data allow the investigators to judge the results objectively. Previous studies by our laboratory,16,21 as well as others, demonstrate concordance between mRNA and protein levels for DPD, TP, and TNF- and suggest that the protein levels would also remain unchanged. However, this method contains some weakness. All of the biopsy specimens taken may not always be tumor tissues, and intratumoral heterogeneity in TP content has been reported.33 Another limitation of these analyses remains the inability for direct confirmation by determination of enzyme activity (DPD and TP) or protein levels (TNF-) is the limited amount of tissue collected from EUS-FNA biopsies. To avoid these biases in sampling, biopsy specimens were obtained from the site of the primary lesion, at which the diagnosis of adenocarcinoma had been confirmed by previous endoscopy by the same gastroenterologist and cancer cells identified by a pathologist, whenever possible.
The major benefit of capecitabine lies in its favorable toxicity profile, oral administration, and, most importantly, its activity in pancreatic cancer both as a single agent as well as a radiosensitizer. Although this first prospective study of capecitabine with radiation in pancreatic cancer recruited a small number of patients, it indicates that the combination can be delivered safely. We did not observe any patient developing grade 3 HFS, the most common DLT observed in rectal cancer studies.34,35 All the patients enrolled on our study were administered pyridoxine 50 mg orally tid36,37 and Udderly Smooth Udder Cream (Reddex Industries Inc, Salem, OH) or Bag Balm (Dairy Association, Lyndonville, VT) bid.38 The major pitfall, as related to any oral agent, is in patients controlling their medication. In this study, only one patient made mistakes in dosing, and was taken off the study because of safety issues. A close diary, in which patients were asked to sign and write down the time they took their capecitabine, was followed on each visit Currently, a phase II study is recruiting more patients at our institution to further confirm the activity of this regimen and to analyze the role of these tumor markers as markers of response to capecitabine.
In conclusion, this hypothesis-driven study demonstrates that capecitabine and concurrent XRT for locally-advanced pancreatic cancer appears safe and well tolerated without unexpected toxicities.
Additionally, capecitabine has the convenience of oral administration and avoidance of catheter-related problems. Tumor response and survival were comparable if not better to standard treatment with infusion FU,4 although to test this vigorously, a phase III trial comparing the two treatment routes is needed. Role of TP and TP:DPD ratio warrants further investigation in a larger clinical trial.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
NOTES
Supported by Grant No. 1 P20 CA101955-01 (Pancreatic SPORE) and a grant from Roche Laboratories.
Presented at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Cartwright TH, Cohn A, Varkey JA, et al: Phase II study of oral capecitabine in patients with advanced or metastatic pancreatic cancer. J Clin Oncol 20:160-164, 2002
Tsukamoto Y, Kato Y, Ura M, et al: A physiologically based pharmacokinetic analysis of capecitabine, a triple prodrug of 5-FU, in humans: The mechanism for tumor-selective accumulation of 5-FU. Pharm Res 18:1190-1202, 2001
Sawada N, Ishikawa T, Sekiguchi F, et al: X-ray irradiation induces thymidine phosphorylase and enhances the efficacy of CAP in human xenografts. Clin Cancer Res 5:2948-2953, 1999
Blanquicett C, Johnson MR, Heslin M, et al: Housekeeping gene variability in normal and carcinomatous colorectal and liver tissues: Applications in pharmacogenomic gene expression studies. Anal Biochem 303:209-214, 2002
Blanquicett C, Saif MW, Buchsbaum D, et al: Antitumor efficacy of capecitabine and celecoxib in irradiated and lead shielded, contralateral human BxPC-3 pancreatic xenografts: Clinical implications of abscopal effects in a metastatic model. Clin Cancer Res (in press)
Eloubeidi MA, Chen VK, Eltoum IA, et al: Endoscopic ultrasound-guided fine needle aspiration biopsy of patients with suspected pancreatic cancer: Diagnostic accuracy and acute and 30-day complications. Am J Gastroenterol 98:2663-2668, 2003
Cancer Therapy Evaluation Program. Common Toxicity Criteria, Version 2.0. Bethesda, MD, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Department of Health and Human Services. March 1998
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Blanquicett C, Gillespie GY, Nabors LB, et al: Induction of thymidine phosphorylase in both irradiated and shielded, contralateral human U87MG glioma xenografts: Implications for a dual modality treatment using capecitabine and irradiation. Mol Cancer Ther 1:1139-1145, 2002
Johnson MR, Wang K, Smith JB, et al: Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem 278:175-184, 2000
Piantadosi S: Clinical Trials: A Methodologic Perspective. New York, NY, John Wiley, 1997, pp 151-155
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ABSTRACT
PURPOSE: To establish the feasibility of capecitabine with concurrent radiotherapy (XRT) in patients with locally advanced (LA) pancreatic cancer and evaluate the effect of XRT on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-alpha (TNF-).
PATIENTS AND METHODS: Fifteen patients with LA pancreatic cancer received three-dimensional conformal XRT to a dose of 50.4 Gy with capecitabine at escalating doses from 600 to 1,250 mg/m2 bid (Monday through Friday). Following chemo-XRT, stable and responding patients were treated with capecitabine 2,000 mg/m2 orally bid for 14 days every 21 days. Tumor specimens were procured with endoscopic ultrasound–guided fine-needle aspiration 1 week before and 2 weeks after chemo-XRT to evaluate TP, DPD, and TNF- mRNA levels.
RESULTS: Dose-limiting grade 3 diarrhea was observed in two of six patients treated at a capecitabine dose of 1,000 mg/m2 with XRT. Three patients (20%) achieved partial response. Mean percent difference in TP pre- and post-XRT was 119.2% (P = .1934). There was no significant differences in mean TNF-, or DPD levels pre- and post-XRT (P = .1934 and .4922, respectively). TP and TNF- levels were not significantly correlated both at pre- and post-XRT (P = .670 and P < .154, respectively). Median value of TP:DPD ratios at baseline was 2.65 (range, 0.36 to 11.08). No association between TP:DPD ratio and efficacy of capecitabine or severity of toxicities was identified.
CONCLUSION: The recommended dose for phase II evaluation is capecitabine 800 mg/m2 bid (Monday through Friday) with concurrent XRT. This approach offers an easy alternative to intravenous fluorouracil as a radiosensitizer in these patients. Role of TP and TP:DPD ratio warrants further investigation in a larger clinical trial.
INTRODUCTION
Pancreatic cancer, one of the most common gastrointestinal tumors, has a 5-year survival of less than 5%.1 Despite representing only 2% to 3% of the total cancer incidence, it is the fourth leading cause of cancer death in the United States.2,3 The majority of patients have advanced disease at the time of diagnosis, and treatment options are limited for this subset of patients. Standard treatment for locally advanced disease remains fluorouracil (FU; either bolus or continuous infusion) and 50 to 60 Gy radiation (XRT).4,5 The schedule involving continuous infusion of FU requires indwelling catheters and ambulatory pumps and is more difficult to administer on an outpatient basis. Although this combined therapy has been shown to increase local control and median survival from 8 to 12 months,4,5 almost all patients succumb to the disease secondary to recurrence, either local or distant. Another more recent method uses gemcitabine as a radiosensitizer, but the optimal dose of gemcitabine with XRT is still not defined, nor is it known whether this regimen would be superior to FU chemoradiotherapy.6 A new promising chemoradiotherapy approach is to combine capecitabine with XRT.
Capecitabine (Xeloda; Hoffmann-La Roche Inc, Nutley, NJ) is an oral fluoropyrimidine that generates FU preferentially at the tumor site by exploiting the higher activity of the enzyme thymidine phosphorylase (TP) in tumor compared with normal tissue.7 The tumor-selective activation of capecitabine potentially enhances safety by minimizing systemic exposure to FU. In clinical trials, capecitabine monotherapy has demonstrated a favorable safety profile that is typical of infused fluoropyrimidines.7,8 The majority of adverse events were mild to moderate in severity, with a low incidence of alopecia and myelosuppression. As an oral agent, capecitabine can be administered in the outpatient setting while, as a result of rapid and almost complete absorption in the upper gastrointestinal tract, providing a pharmacokinetic profile similar to low-dose continuous infusion of FU.8 This offers a more convenient therapy for patients, avoiding the complications and pain associated with intravenous (IV) therapies. Two different administration schedules were explored in phase I studies9,10: the intermittent schedule (2 weeks of treatment followed by 1 week of rest) with a dosage of 1,250 mg/m2 bid, which was used for further phase III development of capecitabine in colorectal and breast cancer; and a 21-day continuous regimen. The latter study reached a maximum-tolerated dose (MTD) of capecitabine 829 mg/m2 bid, and hand-foot syndrome, nausea, vomiting, vertigo, abdominal pain, diarrhea, and thrombocytopenia were reported as dose-limiting toxicities (DLTs).11
Capecitabine is active in pancreatic cancer as a single agent. Cartwright et al12 performed a phase II trial evaluating capecitabine (2,500 mg/m2/d bid x 14 days, every 3 weeks) in patients with advanced pancreatic cancer. Among 38 assessable patients, there were three partial responses, and 11 patients had stable disease for more than four treatment cycles. Median time to progression was 111 days and median survival was 214 days. Clinical benefit response was positive in 17% of patients and stable in 21% of patients. Responses were as follows: 24% positive responses in pain intensity, 12% in analgesic consumption, and 2% in Karnofsky performance status (KPS). Seven of 42 patients had a decrease of 20 points from baseline in their KPS score, with median time to deterioration of 57 days. Weight was maintained within 10% of baseline. The most common grade 3/4 related adverse events were hand-foot syndrome (17%), diarrhea (17%), and nausea (10%).12
As an oral agent that mimics continuous-infusion FU, capecitabine has the potential to replace IV FU and simplify chemoradiotherapy for pancreatic cancer. Earlier preclinical studies using human colon and breast tumor xenograft models suggested that TP and dihydropyrimidine dehydrogenase (DPD) are the best indicators of tumor response to capecitabine with elevated TP expression resulting in higher intratumor levels of FU.8 Conversely, high DPD levels inversely correlate with intratumor FU because of increased degradation.13 Radiation has been shown to significantly increase the efficacy of capecitabine through induction of TP.14 Of particular interest is that the induction of TP by XRT appears to be tumor associated, and this induction of TP may extend to tumors completely outside the field of XRT (abscopal effects).15 Recent pancreatic xenograft studies from our laboratory demonstrated a synergistic antitumor effect with concomitant capecitabine and XRT in both radiated and contralateral lead-shielded tumors in the same animals.16 Interestingly, these studies also suggest that genes other than TP and DPD may be responsible for the synergistic antitumor effect observed with concurrent administration of capecitabine and XRT.16 Taken collectively, these studies have implications in the rational design of treatment paradigms for pancreatic cancer where metastatic disease remains the primary cause of patient morbidity
We report the results of a phase I dose-escalation study conducted to determine the MTD of oral capecitabine administered concomitantly with standard XRT in patients with locally advanced, unresectable pancreatic cancer. To exploit any potential upregulation of TP secondary to XRT, tumor biopsies before and after XRT were performed.
PATIENTS AND METHODS
The study was conducted according to the principles of the Declaration of Helsinki as amended in Somerset West in 1996, and to good clinical practice guidelines. Approval was gained from the institutional review board, and each patient gave written informed consent before being recruited onto the trial.
Eligibility Criteria
Male or female patients 19 years of age with histologically confirmed pancreatic adenocarcinoma determined unresectable by surgeons (on the basis of vascular involvement as per institution's standard) were eligible for this study. Further inclusion criteria were Eastern Cooperative Oncology Group performance status 0 to 2, adequate bone marrow function defined as absolute neutrophil count 1.5 x 103/mm3, platelet count 100 x 103/mm3 and hemoglobin 9.0 g/dL, adequate renal function defined as a serum creatinine < 1.6 mg/dL, adequate hepatic function defined as a serum bilirubin 2 mg/dL and AST levels 2.5x upper limit of normal, and patients capable of providing written consent.
Exclusion criteria included presence of distant metastases, including liver and lungs, previous chemotherapy, XRT or chemoradiotherapy for pancreatic cancer. The following patients were also excluded: pregnant or lactating patients, women with childbearing potential who lacked a reliable contraceptive method, patients with organ allografts, patients with significant cardiac disease, and patients with CNS metastases or a history of uncontrolled seizures, CNS disorders, or psychologic disability thought to be clinically significant, precluding informed consent or adversely affecting patient compliance. Patients were also excluded if they had serious, uncontrolled infections, malabsorption syndrome, or if they lacked physical integrity of the upper gastrointestinal tract ensuring rapid and reproducible absorption of the drug, and patients with known sensitivity to fluoropyrimidines.
Study Design and Treatment
The primary objective of the study was to determine the MTD of oral capecitabine administered continuously bid in combination with standard XRT in patients with locally advanced pancreatic cancer, using a standard escalation design. Secondary objectives included evaluation of the upregulation of TP and tumor necrosis factor-alpha (TFN-), the safety profile, preliminary assessment of the antitumor activity of the combined-modality treatment, and association of TP:DPD ratio to outcome.
Study treatment was started within 14 days after screening assessment. The study consisted of two parts: part A and part B (Fig 1). During part A, patients received concurrent XRT with escalating doses of capecitabine (Monday through Friday) during a period of approximately 6 weeks until they reached the MTD. After finishing part A, patients were assessed for tumor response and/or resectability to potentially undergo a surgical resection. During part B, stable and responding patients were treated with capecitabine 2,000 mg/m2 orally bid for 14 days every 21 days (one cycle) until evidence of progressive disease, unacceptable toxicity, or patient's preference. Restaging was performed every three cycles (9 weeks). Dose escalation in cohorts of three new patients per dose level using a standard phase I study design. Up to six patients were included in the dose cohort if one DLT was observed in any cohort, or if a maximal dose of 2,500 mg/m2/d was reached. Tumor specimens were obtained by a single experienced endosonographer, with endoscopic ultrasound–guided fine-needle aspiration (EUS-FNA) biopsies performed 1 week before and 2 weeks after chemoradiotherapy as previously described.17 Complications were assessed immediately by the endosonographer and at 2 weeks per University of Alabama at Birmingham (UAB) protocol.17
Patients were irradiated once daily, 5 days per week, at 1.8 Gy per fraction, throughout the course of 6 weeks. Computed tomography (CT) image–based three-dimensional treatment planning was utilized to optimize XRT treatment planning by facilitating identification of the target volume and surrounding normal structures (Fig 1). Attempts were made to minimize XRT to surrounding normal tissues while ensuring adequate dose to the target volume. CT simulation was performed with IV and oral contrast material to assist in localizing kidneys, liver, stomach, and intestines. Anatomic structures were contoured for dose-volume histogram (DVH) analysis. The intestines were defined as the contents within the peritoneal cavity, excluding the stomach, spleen, liver, kidneys, aorta, and gross target volume (GTV) to allow for organ motion. The maximum extent of the tumor and involved nodal areas (gross tumor volume), plus adjacent locoregional nodes (ie, celiac, peripancreatic, and portal), and para-aortic nodal areas at risk for residual microscopic diseases (clinical target volume [CTV]) were also defined by CT.
XRT began on the first day of week 1 of the study. The initial target volume received 1.8 Gy/d delivered Monday through Friday for 25 fractions (45 Gy). Typically, the edges of the initial fields were defined superiorly 1.5 cm above the CTV, inferiorly to cover the para-aortic nodes to the L3-4 intervertebral space, laterally and anteriorly with a 1.5-cm margin around the CTV and posteriorly by splitting the anterior vertebral bodies in half. After 45 Gy, an additional three to five 1.8-Gy fractions were delivered to the GTV or tumor bed with a 1.5-cm margin, for a total dose of 50.4 to 54 Gy. XRT was delivered from high-energy linear accelerators with 15-MV photon beams. Patients received 45 to 54 Gy, median dose of 50.4 Gy to the tumor bed.
Dose Modifications
Capecitabine was to be administered at planned escalating doses of 600, 800, 1,000, and 1,250 mg/m2 bid Monday through Friday (weekends off) for the duration of XRT. The first daily dose was administered approximately 2 hours before XRT, with the second dose administered 12 hours after the first. The following recommendations for dose reductions were applied: if a patient experienced a grade 2 or 3 toxicity that was considered possibly related to capecitabine treatment or clearly not related solely to XRT, capecitabine treatment was interrupted and appropriate prophylactic treatment administered. When the toxicity resolved to grade 0 to 1, treatment was resumed without dose adjustment. However, in the case of grade 3 nausea/vomiting or diarrhea not resolving to grade 0 to 1 within 2 days of interruption despite symptomatic treatment, capecitabine treatment was resumed at the preceding dose level without prophylactic treatment. On the recurrence of toxicity at grade 2 or more severe intensity, treatment was interrupted until the toxicity had resolved to grade 0 to 1. Treatment was resumed at the preceding capecitabine dose level (or at the same dose level in patients treated at the lowest dose level). The XRT schedule was not modified unless the severity of the toxicity worsened or a new toxicity of grade 2 or more severe intensity occurred. If a toxicity considered to be clearly related to XRT, such as local skin toxicity, occurred at grade 2 or more severe intensity, XRT was interrupted until the toxicity had resolved to grade 0 to 1 and then resumed with appropriate prophylactic treatment, if required. If the toxicity recurred at grade 2 or more severe intensity, XRT was interrupted until the toxicity had resolved to grade 0 to 1. The administration of capecitabine was not modified unless the severity of the toxicity worsened or a new grade 2 or more severe toxicity developed. If grade 4 toxicities developed, the combination treatment was discontinued, unless the investigator considered it to be in the best interest of the patient to continue treatment with XRT alone or in combination with a reduced dose of capecitabine.
Evaluation of Safety and Efficacy
Adverse events were graded according to National Cancer Institute Common Toxicity Criteria (version 2.0), with the exception of hand-foot syndrome (HFS), which was graded 1 to 3.18 DLT was defined as the occurrence of one or more of the following: any nonhematologic grade 3 toxicity except alopecia, grade 4 vomiting or grade 3 stomatitis, or diarrhea that does not resolve to grade 0 to 2 within 2 days, or grade 3 HFS that does not resolve to grade 0 to 2 within 1 week of starting symptomatic/prophylactic treatment; and grade 4 neutropenia, grade 3/4 neutropenic fever, grade 3 thrombocytopenia, hemorrhage or infection, grade 4 hyperbilirubinemia, or grade 3 shift in liver transaminase concentrations. Adverse events requiring interruption of capecitabine for more than 2 weeks were also classified as dose limiting.
Three patients were recruited to each dose level. The capecitabine dose was escalated when all three patients had completed a minimum of 6 weeks' treatment without DLTs, with at least one patient completing the entire treatment course. If one patient experienced a DLT, an additional three patients were recruited to the same dose level. At dose level three (1,000 mg/m2 bid), six patients were recruited because of grade 3 diarrhea that occurred in one of the first three patients. The MTD was defined as the dose below that caused DLTs in at least two patients in a cohort of six patients.
The safety analysis included all patients who received at least one dose of capecitabine in combination with one dose of XRT. All adverse events were monitored continuously during treatment and for 6 weeks after the end of treatment. Hematology and clinical chemistry was performed weekly during chemo-XRT and thereafter, every 3 weeks during treatment with capecitabine monotherapy. Following chemo-XRT, tumors were evaluated on the basis of Response Evaluation Criteria in Solid Tumors Group 19 criteria at baseline, and 3 to 4 weeks after chemo-XRT.
Tissue Collection, Preparation, and Determination of TP, TNF-, and DPD
Specimens of primary pancreatic ductal adenocarcinoma were obtained via EUS-guided biopsy before starting XRT on day 1 and during week 2 after chemoradiation. Tissues to be utilized for RNA extraction were snap frozen in liquid nitrogen and stored at –80°C. Total RNA was isolated using the Qiagen RNA Purification kit following manufacturer's instruction (Qiagen, Valencia, CA). All sample concentrations were calculated spectrophotometrically at A260 and diluted to a final concentration of 20 ng/μL in RNAse-free water containing 12.5 ng/μL of total yeast RNA (Ambion, Austin, TX) as a carrier.
Real-Time Quantitative Polymerase Chain Reaction
Expression levels were determined using an ABI 7900 Sequence Detection System as previously described by our laboratory.16,20,21 The real-time quantitative polymerase chain reaction (RT-Q-PCR) primers were as follows: human TP forward (5' TCC TGC GGA CGG AAT CC-3'), reverse (5'TGA GAA TGG AGG CTG TGA TGA G-3') and fluorophore-labeled probe (FAM - CAG CCA GAG ATG TGA CAG CCA CCG T-TAMRA); COX-2 forward (5'GAA TCA TTC ACC AGG CAA ATT G-3'), reverse (5'TCT GTA CTG CGG GTG GAA CA-3'), and probe (FAM-TGG CAG GGT TGC TGG TGG TAG GA-TAMRA). The sequence for the primers and probes for human DPD, and S9 ribosomal have been previously described.20,21 Expression levels were calculated using the relative standard curve method.20,21 All reactions were run in triplicate and standard curves with correlation coefficients falling below 0.98 were repeated. Control reactions confirmed that no amplification occurred when yeast total RNA was used as a template or when no-template control reactions were performed.
Statistical Methods
An escalation design with three to six patients was chosen on empiric grounds, according to current standards in phase I cancer trials.22 The chance of not detecting a toxicity that occurs in fact in every second patient is only 1.6% in a cohort of six patients, and less than 0.1% in a cohort of 12 patients. According to the exploratory nature of this pilot trial, only descriptive statistics are presented with respect to response rates, as well as, means with standard deviation (SD), medians and ranges for other quantitative terms. In comparing, pre- and postradiation TP, DPD, and TNF- mRNA levels, the results of a paired t test and signed rank Wilcoxon test are reported. Sample correlation coefficients reported were computed as Pearson's product moment.
RESULTS
A total of 15 patients were accrued onto the study at UAB between April 2003 and March 2004. The demographic characteristics of the patients enrolled are listed in Table 1. The patients had newly diagnosed adenocarcinoma of pancreas, found unresectable on the basis of EUS and CT scan findings as per institutional guidelines. There were no major deviations from the protocol except that one patient (dose level 2) withdrew the consent during therapy, and two others were taken off the study because of reasons unrelated to the study: one patient developed duodenal obstruction and the second patient developed jaundice, work-up of which showed new liver metastases. None of the patients experienced any complications from EUS-FNA after chemoradiotherapy. In particular, none experienced pancreatitis, infection, or hemorrhage.
Dose Escalation and DLTs
Three patients were treated at the lowest dose levels of 600 and 800 mg/m2 bid, respectively, with no DLTs observed. Two of the six patients treated with capecitabine 1,000 mg/m2 bid experienced grade 3 diarrhea, occurring during weeks 4 and 5, respectively (Table 2). Only one patient (1,000 mg/m2 bid) experienced HFS (grade 2) during chemo-XRT. There were no capecitabine dose reductions whatsoever. Apart from the cases mentioned herein and the two patients experiencing DLTs, no capecitabine treatment interruptions were required. Therefore, on the basis of the DLTs, 800 mg/m2 bid Monday through Friday with concomitant XRT is defined as the MTD.
Hematologic Toxicity
No hematologic toxicities except grade 1 anemia were observed during chemo-XRT. All these anemic patients were treated with epoetin alfa without any requirement for blood transfusion. Grade 1 to 2 anemia was also seen during capecitabine alone, not different from that seen in other trials. Leukocytes showed no marked decrease, with the median values staying slightly above the lower limit of the normal range (4.0/nL) throughout the study period. Platelet counts showed a similar decrease, again with no trend toward cumulative toxicity. Counts below the normal range were rare.
Nonhematologic Toxicity
Adverse events according to maximum National Cancer Institute Common Toxicity Criteria are presented for the whole patient group (Table 2). Gastrointestinal toxicities occurred in approximately half of all patients, consisting primarily of grade 1 to 2 nausea (grade 1 in seven patients and grade 2 in three patients) and vomiting (grade 1 in three patients and grade 2 in two patients). Diarrhea occurred in four patients at grade 1, one patient at grade 2, and in two patients at grade 3, the latter being observed at the highest dose level (1,000 mg/m2 bid). According to the study protocol, this event was considered to be a DLT. Grade 1 mucositis was noticed in one patient at 600 mg/m2. Five patients developed grade 1 fatigue and two patients had grade 2 fatigue. Increased values of bilirubin without clinical relevance, common in other capecitabine trials, were recorded in few patients during capecitabine-alone therapy. One grade 2 hyperbilirubinemia at 600 mg/m2 and one grade 1 hyperbilirubinemia at 800 mg/m2 were observed during the chemo-XRT period. Only one of these two patients was found to have metastatic liver disease on evaluation for increased bilirubin. One patient developed grade 2 HFS during week 6, at dose levels of 1,000 mg/m2. No grade 3 HFS was observed during chemo-XRT period. Only one patient developed grade 2 HFS during cycle five of capecitabine monotherapy, which later worsened to grade 3 during cycle eight. Three additional patients had grade 1 HFS. Skin toxicity was observed in approximately 30% of patients (grades 1 and 2), mainly in the irradiated regions and was attributable in most cases to XRT. Also, patients tolerated capecitabine well when administered alone. Except for the observed DLT diarrhea, no clear-cut dose-toxicity relationship was evident.
Antitumor Activity
Among 15 assessable patients, overall response rate was 20%, with three patients achieving a partial response. No patient was found to be resectable because of persistent vascular involvement. Four patients had stable disease after finishing chemo-XRT. Nine patients continued on capecitabine cycles after treatment with concurrent capecitabine and XRT. Median number of cycles administered was six (range, three to 17). Median survival was 14 months, with three patients still surviving at 20 months. Two of these three patients are currently on second-line and third-line chemotherapy, respectively. Most patients received gemcitabine on evidence of progressive disease. Although there was no local disease progression, seven patients developed progressive diseases because of liver metastases.
Quantitation of TP, TNF-, and DPD Expression
TP, TNF-, and DPD mRNA was quantitated in pancreatic tumor tissue by RT-Q-PCR (Tables 3 and 4). The mean percentage of difference in TP expression pre- and post-XRT was 119.2% (median, 61.2%; SD, 175.7%; P = .1934). There was no significant differences in the mean TNF- or DPD levels pre- and post-XRT (P = .1934 and .4922, respectively). TP and TNF- levels were not significantly correlated both at pre- and post-XRT (r = –0.137; P = .670 and r = 0.486; P < .154, respectively). This finding may suggest that mediators other than TNF- may be involved in the upregulation of TP by XRT. The median value of TP:DPD ratios, at baseline, was 2.65 (range, 0.36 to 11.08; Table 5). No association between TP:DPD ratio and efficacy of capecitabine was identified. No correlation was made to overall survival because of the small number of patients. There was no significant correlation between the TP:DPD ratios and the severity of toxicities. None of the patients who developed DLTs were found to be DPD deficient.
DISCUSSION
Two of the six patients treated with capecitabine 1,000 mg/m2 bid experienced grade 3 diarrhea defined as DLT, 1,000 mg/m2 bid Monday through Friday with concomitant XRT was defined as the MTD. Therefore, capecitabine 800 mg/m2 bid Monday through Friday with concomitant XRT is the recommended dosage for subsequent trials.
This is the first human trial in pancreatic cancer that analyzed the intratumor levels of TP and DPD as predictors of tumor response to capecitabine. TP is an enzyme that catalyzes the mutual transformation of the pyrimidine nucleosides thymidine and thymine in nucleic acid metabolism. TP is also an enzyme that converts the FU-based anticancer drugs, 5'-deoxy-5-fluorouridine (5'-DFUR) and its derivative, capecitabine, into FU, and it is, therefore, a limiting factor of the antitumor effects of these anticancer drugs. Capecitabine is a prodrug that is converted to 5'-DFUR in the liver and tumor tissue. Because it is reported that TP is the same protein as platelet-derived endothelial cell growth factor,23 it is considered as a factor involved in tumor angiogenesis and reflects the biologic characteristics of cancer. Prior reports in stomach cancer and colon cancer show that high TP is associated with a poor prognosis,24,25 and that the FU/LV drug regimen is ineffective in colon cancer,26 suggesting that cases of colon cancer with a high TP should be treated with 5'-DFUR and capecitabine. The relationship between the antitumor effects of FU-based anticancer drugs and DPD27 has been noted. Studies on human xenograft models have also shown increased levels of TNF- with increased TP levels14 after XRT. Because TNF- is an upregulator of TP,14 these studies suggest that XRT might increase TP levels indirectly through TNF- in the tumor tissue. Therefore, TP and TNF- expression levels were measured pre- and post-XRT for each patient in our study. The mean difference in TP expression pre- and post-XRT was 119.2 (P = .1934). There was no significant differences in the mean TNF- pre- and post-XRT (P = .1934). And TP and TNF- levels were not significantly correlated both at pre- and post-XRT (r = –.137; P = .670 and r = .486; P < .154, respectively). This finding may suggest that mediators other than TNF- may be involved in the upregulation of TP by XRT.
Recently, preclinical studies on human cancer xenograft models as well as a clinical study using 5'-DFUR in adjuvant chemotherapy in gastric cancer revealed that the efficacy of 5'-DFUR was correlated with the ratio of TP to DPD (TP:DPD) activity.28-30 On the basis of these data, we investigated the relation between TP and DPD expression and the efficacy of capecitabine. No association between TP:DPD ratio and efficacy of capecitabine was identified. There was no significant correlation between the TP:DPD ratios and the severity of toxicities and none of the patients who developed DLTs were DPD deficient.
The results of this phase I study are in accordance with our pancreatic xenograft models demonstrating the synergistic effects of capecitabine with concomitant XRT, but no induction of TP levels in either radiated or contralateral shielded xenografts.16 While these results support the concurrent use of capecitabine and XRT in pancreatic cancer, there appears to be additional genes (other than TP and DPD) associated with response to capecitabine alone and with XRT. The molecular basis of synergy has been reported to be induction of TP expression following XRT.14 Interestingly, our pancreatic xenograft model, which was used to examine response to capecitabine in radiated and contralateral lead–shielded xenografts, demonstrated abscopal effects, with increased antitumor efficacy to capecitabine occurring in tumors outside the field of XRT.16 The precise mechanism(s) by which XRT results in tumor-associated TP induction remains to be elucidated. However, the delay between XRT and the increase in TP expression levels suggests a secondary/mediated induction of TP, rather than a direct effect. Further, the abscopal induction of TP in shielded contralateral xenografts suggests that soluble cell factor(s) may be involved. Initial studies have focused on TNF- and interferon-, which have been shown to both increase after XRT and induce TP expression when added as single agents in vitro.14,31 Taken collectively with analysis demonstrating elevated TP expression in pancreatic tumor compared with normal pancreas, these studies provided the rationale for this phase I clinical study.
There are many methods for the measurement of TP and DPD, including immunohistochemistry, RT-Q-PCR and an enzyme-activity assay.16,20,21,24-26,32 Nishida et al.24 reported that the enzymatic activity of TP closely correlates with the enzyme protein content in the tumor tissues of stomach cancer, colon cancer, and breast cancer (r = .909; P < .001). The immunohistochemical method is the most widely used because of its convenience, but it is difficult to measure the amounts of TP and DPD quantitatively. Instead, we used RT-Q-PCR to measure TP and DPD in this study. This method is also convenient and allows the quantification of enzyme activity in small pieces of tissue such as endoscopic biopsy specimens. These quantitative data allow the investigators to judge the results objectively. Previous studies by our laboratory,16,21 as well as others, demonstrate concordance between mRNA and protein levels for DPD, TP, and TNF- and suggest that the protein levels would also remain unchanged. However, this method contains some weakness. All of the biopsy specimens taken may not always be tumor tissues, and intratumoral heterogeneity in TP content has been reported.33 Another limitation of these analyses remains the inability for direct confirmation by determination of enzyme activity (DPD and TP) or protein levels (TNF-) is the limited amount of tissue collected from EUS-FNA biopsies. To avoid these biases in sampling, biopsy specimens were obtained from the site of the primary lesion, at which the diagnosis of adenocarcinoma had been confirmed by previous endoscopy by the same gastroenterologist and cancer cells identified by a pathologist, whenever possible.
The major benefit of capecitabine lies in its favorable toxicity profile, oral administration, and, most importantly, its activity in pancreatic cancer both as a single agent as well as a radiosensitizer. Although this first prospective study of capecitabine with radiation in pancreatic cancer recruited a small number of patients, it indicates that the combination can be delivered safely. We did not observe any patient developing grade 3 HFS, the most common DLT observed in rectal cancer studies.34,35 All the patients enrolled on our study were administered pyridoxine 50 mg orally tid36,37 and Udderly Smooth Udder Cream (Reddex Industries Inc, Salem, OH) or Bag Balm (Dairy Association, Lyndonville, VT) bid.38 The major pitfall, as related to any oral agent, is in patients controlling their medication. In this study, only one patient made mistakes in dosing, and was taken off the study because of safety issues. A close diary, in which patients were asked to sign and write down the time they took their capecitabine, was followed on each visit Currently, a phase II study is recruiting more patients at our institution to further confirm the activity of this regimen and to analyze the role of these tumor markers as markers of response to capecitabine.
In conclusion, this hypothesis-driven study demonstrates that capecitabine and concurrent XRT for locally-advanced pancreatic cancer appears safe and well tolerated without unexpected toxicities.
Additionally, capecitabine has the convenience of oral administration and avoidance of catheter-related problems. Tumor response and survival were comparable if not better to standard treatment with infusion FU,4 although to test this vigorously, a phase III trial comparing the two treatment routes is needed. Role of TP and TP:DPD ratio warrants further investigation in a larger clinical trial.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
NOTES
Supported by Grant No. 1 P20 CA101955-01 (Pancreatic SPORE) and a grant from Roche Laboratories.
Presented at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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