Provisional IRB approval was first obtained at the principal investigator’s (RRL) American institution following which specific approval was obtained from in-country appropriate IRBs in the Philippines and Vietnam, and at co-investigators’ institution (Indiana University), again following which the principal investigator’s institution provided final approval. The parent clinical trial whose participants were recruited was similarly approved by the foreign and principal investigator’s IRBs.
This ancillary study was begun after the parent clinical trial had accrued 40% of subjects (total accrual =740). Some patients on the parent trial refused participation in the current ancillary study. Thus the patients entering the current study are not representative of all patients entering the parent trial. The parent trial is a randomized clinical trial evaluating the impact of the timing of surgical oophorectomy in the menstrual cycle on outcomes from operable, hormone receptor positive breast cancer. All patients in the trial underwent surgical oophorectomy on the same day as their primary breast surgery, and began tamoxifen 20 mg per day within five days after this.
We have reasons to believe that the compliance with tamoxifen administration (Nolvadex, supplied by AstraZeneca) is high and thus this factor seems less likely to affect the current pharmacogenetic studies. Specifically, patients are expected to return at 3-month intervals to get their drug, which is free. We have logs recording patient collection of tamoxifen medication. The tamoxifen, endoxifen and other metabolite levels in the 224 individual patients studied to date (vide infra) are all such that regular tamoxifen consumption must be occurring. Patients are not taking any drugs believed to interfere with tamoxifen metabolism which issue has confounded reported studies in western populations.
Plasma, leukocyte and serum specimens after four or more months of tamoxifen treatment were obtained in light-shielded tubes from 224 Vietnamese and Filipino patients. The specimens were frozen at −70 degrees C, and shipped frozen in liquid nitrogen canisters to the Indiana University co-investigators. The logistics of sample collection, storage and transport from these foreign countries to the US have been carefully conducted and are validated by the preliminary data we generated. By four months after initiation of tamoxifen, steady-state concentrations of tamoxifen and metabolites are achieved (Jin et al. 2005). Given that the volume of distribution of tamoxifen and metabolites is large, with long half-lives, diurnal and day-to-day variation in these concentrations in an individual patient is minimal (Lash and Rosenberg 2010).
The CYP2D6 gene is highly polymorphic, with over 70 alleles that exhibit functional consequences identified. We have genotyped for CYP2D6 variants relevant to Filipino and Vietnamese populations.
Genomic DNA was isolated from the leukocytes using the QIAamp DNA Blood Maxi Kit (Qiagen, Valencia, CA, USA) and stored at −20°C until use. The DNA concentrations were measured by Quant-iT DNA Broad Range Kit (Invitrogen, Carlsbad, CA, USA). The DNA was diluted to10 ng/μl and used for performing the CYP2D6 genotyping assays. The genotyping was performed for CYP2D6 alleles *2, *3, *4, *5, *6, *10, and *41 using the predeveloped TaqMan Genotyping Assays (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. CYP2D6 is a highly polymorphic gene with more than 70 different allelic variants. We chose these star alleles because they are more frequent in the Asian and Caucasian population (Bradford 2002; Sistonen et al. 2007; Veiga et al. 2009).
Tamoxifen and its metabolites were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) as described elsewhere (Irvin et al. 2011). Standard curves and quality controls were run with each sample batch. The intraday coefficient of variance was lower than 10% for all compounds, while the inter-day was lower than 15%. An intra-patient assay was not run for each sample set, but repeated measurement was performed in a subset of samples. The results show that the intra-sample variability was lower than 10%.
We have used our preliminary data to explore the relationship between endoxifen concentrations and recurrence prior to follow-up being complete on all patients in our adjuvant trial. In this effort, we designed a nested case–control study. Our rationale was that using such a small case–control training data set might allow us to validate any important trend in a larger, independent data set collected from our trial patients who will have longer follow-up time in the future (Pepe et al. 2008). Among the 224 studied patients we identified 24 patients who had recurrence, and matched them with 24 non-recurrence controls who were from the same enrollment hospital, were of the same disease stage (I-II vs. III), and had a similar enrollment date. An optimal matching technique was utilized, as implemented by Bergstrahl (Bergstralh et al. 1996) and the follow-up time for the control in each pair was required to be at least as long as the time to recurrence for the case.
The effect of CYP2D6 genotype metabolic status on each of the metabolites (tamoxifen, NDMTAM, endoxifen, and 4-OHTAM) was assessed using a test for linear trend. The proportion of variance explained by the linear trend model (r-squared) was also calculated.