Patient selection
This study was performed as a site study of a phase III trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced NSCLC patients [10]. Patients had to meet the inclusion and exclusion criteria of the main study. In short, patients were included if they had stage III/IV NSCLC. No prior chemotherapy was allowed. Radiotherapy should have been completed at least 4 weeks before inclusion, and patients should have recovered from any toxic side effect. All patients had to have a performance status ≤ 2 according to the Eastern Cooperative Oncology Group (ECOG) scale and a life expectancy of at least 12 weeks. An adequate bone marrow reserve and normal renal (creatinine clearance ≥ 60 ml/min) and liver function were required. The glomerular filtration rate (GFR) was calculated according to the formula of Cockcroft and Gault [11]. Patients were excluded if they had active infections, second primary malignancies, uncorrected hypercalcemia, or an LVEF ≤ 45% measured by multiple gated acquisition (MUGA) scan. A detailed description of the inclusion and exclusion criteria is published elsewhere [10]. The local medical ethics committee of the hospital approved the protocol. All patients gave written informed consent before study entry including genotyping procedures.
Treatment and sample collection
Gemcitabine (Gemzar®, Lilly, Nieuwegein, the Netherlands) in a dose of 1,125 mg/m2 in 250 mL 0.9% NaCl solution was administered intravenously as a 30 min infusion. Patients co-treated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion. The cisplatin dose was administered the day after the gemcitabine infusion.
Blood sampling
Blood sampling was carried out on the first day of the first chemotherapy cycle. For pharmacokinetic sampling, a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration. Blood samples of 9 mL were collected in heparinized tubes containing 0.25 mg tetrahydrouridine (THU) in 50 μL water, just before chemotherapy and at t = 25, 40, 50, 60, 75, 90, 105, 120, 150, 180, 270, and 360 min after the start of the gemcitabine infusion. Blood samples were immediately placed on ice and centrifuged at 2,500 g for 10 min within 1 h after collection. Plasma samples were stored at −80°C until further analysis.
Genotyping material
Harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30 s with 5 mL 0.9 % NaCl solution. The cell suspension was collected in plastic cups and subsequently placed on ice. Within 1 h after collection, the cell suspension was centrifuged for 10 min at 190 g at 4°C. The supernatant was discarded and cells were washed in 10 mL ice cold phosphate buffered saline, pH = 7.40 (PBS) and centrifuged for 10 min at 190 g at 4°C. The pellet was resuspended in 1 mL ice cold PBS. The remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly (15 s) at 10,000 g. The supernatant was discarded, and the pellet was kept frozen at −80°C until further analysis.
Pharmacokinetic analysis
Gemcitabine hydrochloride and dFdU were obtained from Eli Lilly (Indianapolis, IN). Tetrahydrouridine was purchased from Calbiochem (La Jolla, CA). All other chemicals were of standard analytical grade. The analysis of gemcitabine and dFdU in plasma was carried out by high performance liquid chromatography as described by Freeman et al. [12].
Cytidine deaminase genotyping
The CDA exon 1 polymorphism (C/A) at codon position 27 was genotyped by direct sequencing, in both directions, of PCR-amplified genomic DNA.
First, the CDA region flanking the polymorphic site was amplified using PCR with forward primer 5′- AGTAGCTTCCCCTTCCAGTAGC and reverse primer 5′-CCTCTTCCTGTACATCTTCCTCTG. The 25 μL reactions contained 2.5 units Taq polymerase (Amersham Biosciences, Uppsala, Sweden); 0.5 mM dNTP mix (Roche Diagnostics, Mannheim, Germany); 1x PCR buffer (Roche Diagnostics), 0.05 mM MgCl2; 0.2 μM of each primer; and approximately 50 ng genomic DNA. The amplification was performed on a PTC-225 thermal cycler (MJ Research, Waltham, MA), using a stepdown protocol. The first five cycles were carried out at 94°C, 65°C, and 72°C, each for 30 s. The next five cyles were carried out at 94°C, 63°C, and 72°C, each for 30 s. The last 25 cyles were at 94°C, 60°C, and 72°C, each for 30 s. Following cycling the PCR products were purified with the Qiagen Qiaquick PCR purification kit (Westburg, Leusden, the Netherlands). Subsequently, 100 ng of the purified PCR product was cycle sequenced with a Dyeterminator kit (US81090, Amersham Biosciences, Roosendaal, the Netherlands) in a thermal cycler (MJ Research), using 0.05 mM sequencing primer. For the reverse reaction, the same primer was used as in the PCR, but for the forward reaction an internal primer was used (5′-GGTACCAACATGGCCCAGAAG). After the cycle reaction the sequencing products were cleaned on a Sephadex plate (Amersham Biosciences) by centrifugation for 5 min at 910 g. The eluted sample was vacuum dried for 45 min at 65°C. Finally, 20 μL of loading solution (Amersham Biosciences) was added to dissolve the sequencing products. The samples were analyzed on a MegaBACE 1000 capillary sequencer (Amersham Biosciences) by injecting the samples for 45 s at 3 kV and running them for 5 h at 4 kV. The data were processed with Sequence Analyzer 3.0 (Amersham Biosciences) and Seqman II (DNASTAR, Madison, WI).
Pharmacokinetic modelling
Pharmacokinetic data were analyzed with the Mw\Pharm software package (version 3.5; MediWare, Groningen, the Netherlands) using the KinFit module. For gemcitabine and its metabolite dFdU, the AUC (using the trapezoid rule), clearance, distribution volume, and elimination half-lives were calculated by non-compartmental analysis.
Statistical analysis
Patient data were grouped according to genotype. Differences in laboratory and demographic data between groups were tested using the Mann-Whitney test.
CDA genotype-related differences in pharmacokinetic data were investigated by analyzing the wild type AA genotype patient group against the combined heterozygote AC and homozygote CC genotype patient group using the Mann-Whitney test. We assumed an effect of 30% of the alternative genotype on the gemcitabine AUC as clinically relevant. From the literature, the allele frequency of the wild type genotype was estimated at 0.7, and therefore an almost equal distribution of patients in AA wild type (49%) and combined homozygote CC plus heterozygote AC (51%) patient groups was expected. We calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean AUC of the mutant group is 30% larger, with group standard deviations of 20% and with a significance level of 0.05 using a two-side Mann-Whitney test assuming that the actual distribution is logistic.
Additionally, differences between uncombined CDA genotype groups regarding gemcitabine AUC, clearance, and half-life were also analyzed using the Kruskall-Wallis test. Statistical analyses were performed using the SPSS 14.0 statistical package (SPSS 2005, Chicago, IL). Power calculations were performed using NCSS 2004 Statistical & Power Analysis Software (NCSS, Kaysville, UT).