We performed a retrospective cohort study utilizing genomic DNA derived from hematologic circulating or bone-marrow-derived stem cells and clinical data from breast cancer patients enrolled on Intergroup Trial 0121 (E2190/SWOG9061/CALGB 9496), a multicenter trial of high dose vs. standard dose adjuvant chemotherapy. Patients were included in the current study if they were enrolled in INT-0121, informed consent was confirmed, and archival peripheral blood or bone marrow stem cells were available for genomic DNA extraction and subsequent genotyping. This study is in compliance with the Helsinki Declaration and was performed with the approval of the University of Pennsylvania Institutional Review Board and the Eastern Cooperative Oncology Group (ECOG) Executive Committee.
Results of E2190/INT-0121 trial have been published previously [9]. Briefly, 540 patients with ≥10 positive lymph nodes received conventional adjuvant therapy with four cycles of cyclophosphamide (C; 100 mg/m2, orally, Days 1 to 14), doxorubicin (A; 30 mg/m2, intravenously, Days 1, 8), and fluorouracil (F; 500 mg/m2, intravenously, Days 1, 8) followed by randomization to either observation or receipt of high-dose chemotherapy (HDC: cyclophosphamide (6 gm/m2) and thiotepa (800 mg/m2) over a four-day period) with hematopoietic stem cell reinfusion. Adjuvant tamoxifen was recommended for patients with estrogen receptor (ER) positive tumors, although receipt of this medication was not tracked in the study database.
The protocol specified hematopoietic stem cell collection at the completion of standard CAF for all patients on the study. Specimens not utilized for autologous reinfusion were stored at -80 C at the ECOG Pathology Core Facility (Chicago, IL, USA). The original INT-0121 consent form included language specifying that residual biological specimens would be used for future breast cancer research. The ECOG Statistical Center (Boston, MA, USA) performed additional follow-up and de-linked patient identifiers from the clinical data used in this analysis. The primary endpoint for this study was DFS, defined as time from randomization to earliest recurrence, new breast cancer, or death. The secondary endpoint was overall survival, defined as time from randomization to death [9]. All survival times were censored at time of last contact or on 1 August 2005 if subjects were alive and disease-free at that time.
We selected 15 SNPs (CYP2B6*4, CYP2B6*5, CYP2B6*6, CYP2B6*7, CYP2B6*9, CYP2C9*2, CYP2C9*3, CYP3A4*1B, CYP3A5*3, CYP3A5*6, CYP2D6*4, GSTM1, GSTT1, GSTP1*B, GSTP1*C) in eight genes for inclusion by first identifying cyclophosphamide-metabolizing enzyme polymorphisms that were associated with functional effects on enzyme expression, levels or activity, then excluding those in which the expected prevalence of the combination of alleles for a particular gene was ≤ 10% of the general population, since these constituted a minute fraction of cyclophosphamide metabolism and significant effects for these SNPs were unlikely to be detectable with the sample size available. All variants were hypothesized to result in decreased enzyme function [10]. Of note, genotyping for CYP2D6*4 was included to account for any possible differential effect of variable tamoxifen metabolism on outcome among patients whose tumors were estrogen-receptor positive.
The ECOG Pathology Core Facility at Northwestern University extracted DNA from hematologic stem cells with the EZ1 system (Qiagen, Inc, Hilden, NW, Germany). Genotyping was performed at the University of Pennsylvania. GSTM1 and GSTT1 homozygous null mutations were detected using a method previously described, using a 4% metaphor agarose gel [11, 12]. The remainder of genotyping was determined by PyroSequencing (CYP2C9*3, CYP3A4*1B, CYP3A5*3, CYP3A5*6, GSTP1*B, GSTP1*C) (Biotage, Charlottesville, VA, USA, Applied Biosystems, Foster City, CA, USA)) and Taqman Real-Time PCR assays on the MJ Research Chromo4 (Bio-Rad) platform (CYP2B6*4, CYP2B6*5, CYP2B6*6, CYP2B6*7, CYP2B6*9, CYP2C9*2, CYP2D6*4). The technician performing genotype assays was blinded to all clinical and outcome data.
Polymorphisms were examined individually and in genotype groups defined based on our prior work [6]. With regard to CYP2B6, assignment of genotype was carried out as follows: carriers of the A785G mutation alone were designated CYP2B6*4; carriers of the C1459T mutation alone were designated CYP2B6*5; carriers of the combination of A785G and G516T were designated CYP2B6*6; carriers of the combination of A785G, G516T, and C1459T were designated CYP2B6*7; and carriers of the G516T mutation alone were designated CYP2B6*9 (data not shown). Because of the similarly anticipated directions of effect and the lack of power to perform comparisons with each polymorphism, dichotomous CYP2B6, CYP2C9, and GSTP1 variables were created based on genotype, where groupings consisted of all wild-type versus carriers of any variant.
To validate our pilot work assessing combined effects of variants in both activating (CYP) and metabolizing (GST) enzymes, we also classified subjects into three groups (favorable, intermediate and unfavorable) based on their CYP3A4*1B, CYP3A5*3, GSTM1 and GSTT1 genotypes [6]. The favorable group was comprised of subjects with no variant in either CYP3A4 or CYP3A5 and null at both GSTM1 and GSTT1. The unfavorable group was comprised of subjects who were variant in either CYP3A4 or CYP3A5 and non-null at both GSTM1 and GSTT1. The intermediate group comprised all other CYP/GST combinations. The groups were hypothesized to have varying serum concentrations of active cyclophosphamide metabolites based on known functional significance of the genetic variants.
STATA (Release 9, Stata Corporation, College Station, TX, USA) and R (Version 2.3.1, R Foundation for Statistical Computing, Vienna, Austria) software were used for statistical analysis. Pearson's chi-squared or exact tests for small samples were used to compare proportions. Survival curves were generated using the Kaplan-Meier method [13] and were compared using the log-rank test. Cox regression models were computed to determine the hazard ratio associated with each genotype, genotype group or clinical variable and both DFS and OS. Indicator variables for categorical variables included a category for missing data. The Grambsch-Therneau test was used to test the proportional hazards assumption [14]. Multivariable Cox models for DFS and OS were developed by including all genotype and clinical variables. All tests of significance were two-sided, with alpha = 0.05.