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Published in final edited form as: Pharmacogenomics J. 2016 Nov 15;18(1):35–42. doi: 10.1038/tpj.2016.75

ABC Transporter Polymorphisms are Associated with Irinotecan Pharmacokinetics and Neutropenia

Megan Li 1, Eric L Seiser 2, R Michael Baldwin 1, Jacqueline Ramirez 3, Mark J Ratain 3, Federico Innocenti 2, Deanna L Kroetz 1,4
PMCID: PMC5432414  NIHMSID: NIHMS837275  PMID: 27845419

Abstract

Neutropenia is a common dose-limiting toxicity associated with irinotecan treatment. While UGT1A1 variants have been associated with neutropenia, a fraction of neutropenia risk remains unaccounted for. To identify additional genetic markers contributing to variability in irinotecan pharmacokinetics and neutropenia, a regression analysis was performed in 78 irinotecan-treated patients to comprehensively analyze three hepatic efflux transporter genes (ABCB1, ABCC1 and ABCG2). rs6498588 (ABCC1) and rs12720066 (ABCB1) were associated with increased SN-38 exposure, and rs17501331 (ABCC1) and rs12720066 were associated with lower absolute neutrophil count nadir. rs6498588 and a variant in high linkage disequilibrium are located in transcriptionally active regions or are predicted to alter transcription factor binding sites. While enhancer activity was not evident in vitro for genomic regions containing these SNPs, rs6498588 was significantly associated with ABCC1 expression in human liver. These results suggest that genetic variation in ABCC1 and ABCB1 may contribute to irinotecan-induced neutropenia by altering expression of transporters involved in irinotecan metabolite disposition.

Introduction

Irinotecan is an antineoplastic prodrug approved for first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic carcinoma of the colon or rectum and as a single agent for second-line therapy of fluorouracil refractory metastatic colorectal cancer. Common dose-limiting toxicities are diarrhea and neutropenia1, with up to 34% of patients experiencing grade 3–4 neutropenia2. Interindividual variation in irinotecan toxicity has prompted the search for genetic biomarkers to guide safer irinotecan treatment.

Irinotecan is activated to form the more potent topoisomerase I inhibiting active metabolite, SN-383. Elevated SN-38 plasma concentrations have been associated with neutropenia during irinotecan treatment4–7. SN-38 is subsequently inactivated to SN-38 glucuronide (SN-38G) by members of the uridine diphosphate glucuronosyl transferase family, primarily UGT1A18 (Figure 1).

Figure 1. Role of ABC transporters and UGT in irinotecan pharmacokinetics and toxicity.

Figure 1

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The pathway is taken from PharmGKB. Copyright PharmGKB. Permission has been given by PharmGKB and Stanford University. An original version is available online at https://www.pharmgkb.org/pathway/PA2001.

UGT1A1*28 has been associated with increased risk of neutropenia during irinotecan treatment5,9–12. The TA repeat polymorphism is located in the proximal promoter region of UGT1A1 and results in reduced expression relative to the UGT1A1*1 reference allele. Patients homozygous for UGT1A1*28 glucuronidate SN-38 less efficiently than patients who have one or more reference alleles10; subsequently, homozygous patients are especially susceptible to toxicities resulting from increased SN-38 systemic exposure (as measured by area under the plasma concentration-time curve (AUC)). UGT1A1*93 (rs10929302), a promoter polymorphism in high linkage disequilibrium (LD) with UGT1A1*28, has also been proposed as a predictor of neutropenia, with the variant being strongly associated with increased hematologic toxicity13, increased SN-38 exposure, and lower absolute neutrophil count (ANC) nadir14–16.

While UGT1A1*28-guided irinotecan dosing does reduce dose-limiting toxicities17, variability in SN-38 AUC within genotype-normalized doses suggests that UGT1A1*28 does not account for all the toxicity observed during irinotecan therapy. Other studies have demonstrated more modest effects of UGT1A1 variants and propose that other metabolizing enzymes and drug transporters may be involved18–20.

Irinotecan toxicity and increased SN-38 exposure have been previously associated with several hepatic transporter polymorphisms14,21–23. The ATP-binding cassette (ABC) family includes proteins that transport irinotecan and its metabolites out of the cell24. ABCB1 variants have been associated with increased exposure to irinotecan and SN-3821, reduced irinotecan and SN-38 clearance21,25, and other toxic events15,26. ABCC1 variants were previously associated with ANC nadir, SN-38 AUC, and the ratio of SN-38G AUC to SN-38 AUC14, and a common ABCG2 haplotype was also associated with neutropenia27. However, these associations have yet to be replicated and validated. Compared to UGT1A1, hepatic transporters are relatively unexplored yet could play an important role in the distribution and accumulation of irinotecan and its metabolites.

In the current study, a candidate gene and functional analysis were performed to examine associations of ABCB1, ABCC1, and ABCG2 polymorphisms with irinotecan pharmacokinetics and neutropenia. An earlier pharmacogenetic analysis in this cohort included only a small number of polymorphisms in these three genes14. The present study covers the entire length and flanking regions of these genes for a more comprehensive analysis and tests the hypothesis that ABC transporter variants can contribute to additional variability in neutropenia observed during irinotecan treatment.

Subjects and Methods

Subjects

Human investigations were performed after review and approval by the Biological Sciences Division/University of Chicago Hospitals Institutional Review Board and in accordance with Federalwide Assurance for the protection of human subjects. Informed consent was obtained from all subjects. The study cohort consisted of 85 advanced cancer patients treated with single-agent irinotecan (300 mg/m2 or 350 mg/m2) every 3 weeks. Pharmacokinetic and clinical data, including race, sex, age, body surface area (BSA), SN-38 AUC, ANC nadir, and baseline ANC, were collected. This patient population was examined in previous studies5,14, and thus the sample size was not based on a priori calculation.

Genotyping

Tag single nucleotide polymorphisms (SNP) were selected from genomic regions covering 25 kb upstream of the transcription start site to 5 kb downstream of the 3′-UTR of each candidate gene (ABCB1, ABCC1, ABCG2). Additional SNPs were chosen to directly interrogate nonsynonymous SNPs in the UCSF Pharmacogenetics of Membrane Transporters database (http://pharmacogenetics.ucsf.edu/), SNPs with published clinical associations, and SNPs in LD with clinically associated SNPs. Fifty ABCB1 SNPs, 93 ABCC1 SNPs, and 38 ABCG2 tag SNPs were assayed. Genotyping was performed by the Mayo Clinic Genotyping Shared Resource using the Illumina VeraCode (BeadXpress) platform. Following quality control measures to eliminate suboptimal samples and markers and filtering for minor allele frequency ≥ 0.05 using PLINK v1.0728, 85 individuals and 123 SNPs were retained for analysis (Supplementary Table 1).

Statistical analysis

SNPs were tested for deviation from Hardy-Weinberg equilibrium using an exact test. Genotype-phenotype associations were analyzed using a two-stage regression analysis in R v3.2.229. Dose-adjusted SN-38 AUC and ANC nadir values were log10-transformed for the analysis. In the univariate analysis, simple regressions were first performed on each variant and clinical covariate separately, and those with a significance level of P < 0.15 were kept for further analysis. Step-wise backward selection of this subset of genotypes using the rms package30 for R led to the final multivariable models, and model fit was assessed by examining R2 values. An additive genetic model was first assumed for all genotype associations. To eliminate the driving effect of any sparse homozygous variants, when a particular variant had n ≤3 observations in the homozygous genotype group and was significant, these genotypes were grouped with the heterozygous genotypes; the variant was kept in the model if still significant after combining genotypes.

Other statistical analyses were performed using GraphPad Prism v6.0 (GraphPad Software, La Jolla, CA, USA). Comparisons for the risk alleles analysis and luciferase reporter assays were analyzed using a Mann-Whitney test or, if there were more than two groups, a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Variance was assumed to be similar between compared groups, and two-sided P-values < 0.05 were considered statistically significant.

Bioinformatic analysis

Significant SNPs and SNPs in high LD (r2 ≥ 0.8) were examined for evidence of changes in function and regulatory activity. HaploReg31 and RegulomeDB32 were used to determine whether the SNPs overlap with experimentally predicted functional elements (chromatin state segmentation33, DNase hypersensitivity peaks, ChIP-seq peaks) from the ENCODE Project34 and the Roadmap Epigenomics Project35. HaploReg31 and TRANSFAC Match36 were used to identify and predict changes in transcription factor (TF) binding.

Measurement of enhancer activity in vitro

Primers were designed with the aid of Primer337,38 to target 500 bp genomic regions flanking each significant SNP (Supplementary Table 2). These regions were PCR-amplified using PfuTurbo (Agilent, Santa Clara, CA, USA) from human genomic DNA and cloned into the pGL4.23[luc2/minP] luciferase reporter vector (Promega, Madison, WI, USA). The QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) was used to create the desired point mutation. Correct sequences were verified by Sanger sequencing. Reporter plasmids containing reference or variant sequences were transfected into Hep G2 (ATCC® HB-8065™, human hepatocellular carcinoma, passage 2–6) cells using Lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA, USA). The Renilla luciferase construct pGL4.73 (Promega) was co-transfected to correct for transfection efficiency. Empty pGL4.23 vector and a known apolipoprotein E liver enhancer39 were also transfected as negative and positive controls, respectively. Cells were cultured in Eagle’s Minimum Essential Medium with EBSS, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, and 10% fetal bovine serum. Twenty-four hours after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) and quantified using the GloMax-96 Microplate Luminometer (Promega). Background-subtracted firefly luciferase activity was normalized with respect to Renilla luciferase activity and expressed as a mean value relative to empty pGL4.23 vector.

Code availability

The R code for the univariate and multivariate regression analyses is available at https://github.com/kroetzlab/irinotecan-ABC.

Results

Cohort analysis

The original study cohort consisted of 85 patients, with the majority being Caucasian or African American. Seven subjects (2 Asian, 1 Filipino, 4 Hispanic) were omitted from the initial regression analyses to avoid dilution of association signals by genetic heterogeneity. Characteristics of patients included in the primary analysis are listed in Supplementary Table 3. The analyzed cohort had a median age of 57 and slightly more males (58%). The majority (76%) received a 350 mg/m2 dose of irinotecan, while 24% received a 300 mg/m2 dose. The outcomes examined in this study were SN-38 AUC (adjusted for irinotecan dose) and ANC nadir. Distributions of these phenotypes in the analyzed cohort are shown in Supplementary Figure 1. None of the clinical covariates (race, sex, age, BSA, baseline ANC) were significantly associated with SN-38 AUC or ANC nadir.

Association of ABC SNPs with SN-38 exposure

Eight SNPs met the P < 0.15 threshold in univariate regression analyses for SN-38 exposure. Of these, rs12720066, rs6498588, rs35621, and rs2373586 had P-values < 0.05 (Table 1). The genotype frequencies for these SNPs in Caucasians and African Americans are shown in Supplementary Table 4. All eight ABC SNPs were taken into the multivariate analysis along with the UGT1A1*93 genotype (rs10929302). The final log SN-38 AUC model included UGT1A1*93, an intronic ABCB1 variant (rs12720066), and an ABCC1 variant (rs6498588) in the 5′-flanking region (Table 1). The model explained 34% (R2 = 0.345) of the variation in SN-38 exposure, with all three SNPs contributing similar variances to the model (rs12720066: β = −0.337; rs6498588: β = 0.325; rs10929302: β = 0.390). The UGT1A1 and ABCC1 SNPs were associated with increased exposure of patients to SN-38, while the ABCB1 SNP was associated with decreased exposure (Figure 2).

Table 1.

Univariate and multivariate analysis results for log SN-38 AUC1

Univariate Multivariate3
rsid Gene Function Ref>Alt Estimate SE P Estimate SE P
rs127200662 ABCB1 intronic A>C −0.204 0.070 0.005 −0.217 0.061 6.24 × 10−4
rs6498588 ABCC1 5′-gene A>T 0.111 0.042 0.010 0.123 0.036 9.50 × 10−4
rs356212 ABCC1 intronic C>T 0.191 0.083 0.025
rs2373586 ABCB1 intronic A>C 0.086 0.040 0.033
rs8187843 ABCC1 intronic G>A −0.160 0.084 0.061
rs37435272 ABCC1 3′-UTR C>T 0.113 0.062 0.071
rs2150952 ABCC1 intronic G>A 0.118 0.073 0.109
rs212082 ABCC1 intronic A>G −0.080 0.054 0.142
rs10929302 UGT1A1 promoter G>A 0.145 0.042 0.001 0.154 0.037 9.00 × 10−5
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1

SN-38 AUC expressed in units of ng*hr/mL; adjusted for irinotecan dose

2

Homozygous alternate genotypes grouped with heterozygous genotypes

3

R2 = 0.345

Figure 2. Association of ABC and UGT variants with SN-38 AUC.

Figure 2

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The relationship between genotype and SN-38 AUC (adjusted for irinotecan dose) is shown for all analyzed patients. Horizontal bars represent the median values for each genotype group.

Inclusion of only Caucasian genotypes in the association analyses yielded similar results as obtained with the combined population (Supplementary Table 5). The multivariable model for log SN-38 AUC also contained UGT1A1*93, rs12720066, and rs6498588, with similar effect sizes as those for the model including African Americans. Inclusion of the seven Asian, Filipino, and Hispanic subjects into the final model (Supplementary Table 6) resulted in similar coefficients and P-values.

Association of ABC SNPs with neutropenia

In univariate linear regression analyses of ABC SNPs with ANC nadir, ten SNPs met the P < 0.15 threshold and three (rs17501331, rs12720066, and rs3743527) had P-values < 0.05 (Table 2). Genotype frequencies of these SNPs are listed in Supplementary Table 4. The final model following multivariable regression and inclusion of UGT1A1*93 included an intronic SNP in ABCB1 (rs12720066) and an intronic SNP in ABCC1 (rs17501331) (Table 2). This model accounted for 39% (R2 = 0.390) of the variation in ANC nadir, with UGT1A1*93 explaining more variance in the outcome (β = −0.479) than the other two SNPs (rs17501331: β = −0.295; rs12720066: β = 0.286). The ABCB1 SNP was associated with increased ANC nadir and the UGT1A1 and ABCC1 SNPs were associated with reduced nadirs (Figure 3). As for the SN-38 AUC analysis, the final multivariable model for log ANC nadir for Caucasians only contained the same SNPs with similar effect sizes as those for the model including African Americans (Supplementary Table 7). Inclusion of the seven Asian, Filipino, and Hispanic subjects into the final model (Supplementary Table 8) also resulted in similar coefficients and P-values.

Table 2.

Univariate and multivariate analysis results for log ANC nadir1

Univariate Multivariate3
rsid Gene Function Ref>Alt Estimate SE P Estimate SE P
rs17501331 ABCC1 intronic A>G −0.255 0.106 0.019 −0.281 0.088 0.002
rs127200662 ABCB1 intronic A>C 0.227 0.102 0.030 0.261 0.084 0.003
rs37435272 ABCC1 3′-UTR C>T −0.194 0.089 0.033
rs212090 ABCC1 3′-UTR T>A 0.102 0.059 0.089
rs1128503 ABCB1 synonymous A>G −0.097 0.056 0.090
rs8050881 ABCC1 5′-gene A>G −0.098 0.061 0.110
rs2725264 ABCG2 intronic C>T −0.127 0.080 0.118
rs35626 ABCC1 intronic G>T −0.107 0.069 0.123
rs1967120 ABCC1 intronic G>A −0.108 0.070 0.124
rs152023 ABCC1 intronic C>T −0.093 0.063 0.144
rs10929302 UGT1A1 promoter G>A −0.262 0.057 1.98 × 10−5 −0.268 0.052 1.99 × 10−6
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1

ANC nadir expressed as cells/μL

2

Homozygous alternate genotypes grouped with heterozygous genotypes

3

R2 = 0.390

Figure 3. Association of ABC and UGT variants with ANC nadir.

Figure 3

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The relationship between genotype and absolute neutrophil count nadir is shown for all analyzed patients. Horizontal bars represent the median values for each genotype group.

Contribution of multiple risk alleles

To examine the combined effects of risk alleles, composite genotypes were constructed for each outcome. Patients carrying more risk alleles for rs12720066, rs6498588, and UGT1A1*93 had significantly higher SN-38 AUC (P < 0.0001), while patients carrying more risk alleles for rs12720066, rs17501331, and UGT1A1*93 had significantly lower ANC values (P = 0.0006) (Supplementary Figure 2). Almost all (91%) patients with grade 3–4 neutropenia (< 1000 cells/μl) carried at least four risk alleles among all three ABC SNPs and UGT1A1*93, compared to 53% of patients with no or less severe neutropenia (P = 0.001). Of the patients with grade 3–4 neutropenia, 91% are homozygous for rs12720066 and 46% are homozygous for UGT1A1*93, while of patients with no or less severe neutropenia, 73% are homozygous for rs12720066 and 7% are homozygous for UGT1A1*93. Dose-adjusted SN-38 AUC was also confirmed to be higher in patients with grade 3–4 neutropenia (Supplementary Figure 3; P = 0.0001).

Bioinformatic analysis

To investigate the putative function of the SNPs in the final models, overlapping regulatory elements and protein binding sites were examined. The intronic ABCC1 SNP rs17501331 is in a predicted region of transcriptional transition in Hep G2 cells and is predicted to alter two regulatory motifs, DMRT1 and SOX6. rs6498588, located 11 kb upstream of ABCC1, is predicted to alter three regulatory motifs (GR, Mef2, YY1). rs4148330, a SNP in high LD (r2 = 0.8) with rs6498588, resides 1.7 kb upstream of ABCC1 in a region with H3K4 methylation in Hep G2 cells and DNase hypersensitivity in Hep G2 cells and hepatocytes, and is predicted to alter E2F and NRSF TF binding. rs12720066 and other SNPs in high LD with rs6498588 were also predicted to alter regulatory motifs but were not associated with an enrichment in enhancer or DNase activity in liver or liver-derived cells.

Enhancer assays

Genomic regions containing the ABCB1 and ABCC1 SNPs in the final models for SN-38 AUC and ANC nadir plus additional SNPs in high LD with these variants (Supplementary Table 2) were tested in vitro for enhancer activity and the effect of genetic variation on this activity. None of the tested genomic regions exhibited significant enhancer activity and the introduction of the indicated variants did not change the transcriptional activity (Figure 4).

Figure 4. Analysis of genomic regions and genetic variants for enhancer activity.

Figure 4

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Enhancer activity was measured in Hep G2 cells using a luciferase reporter gene assay. The values plotted represent mean ± SD of the relative luciferase activity in cells transfected with the indicated genomic region without (Ref) and with (SNP) the variant compared to empty pGL4.23 construct (−). A known liver enhancer of the APOE gene was used as the positive (+) control. Data are representative of at least three independent experiments of six biological replicates each.

Discussion

This study provides a comprehensive analysis of the genetic variants spanning three hepatic ABC transporter genes of the irinotecan pharmacokinetic pathway. Three SNPs (rs12720066, rs6498588, rs17501331) were significantly associated with changes in SN-38 AUC or ANC nadir. These variants could potentially predict neutropenia during irinotecan treatment and provide additional insight into the molecular mechanism of the toxicity.

UGT1A1*93 (rs10929302) was confirmed as a strong predictor of irinotecan-induced neutropenia, reflected by associations with higher SN-38 AUC and lower ANC nadirs. The effect sizes of UGT1A1*93 for associations with SN-38 AUC or ANC nadir were similar or slightly more significant than those of UGT1A1*28. UGT1A1*93 was previously associated with increased hematologic toxicity, increased SN-38 exposure, and lower ANC nadir13–15. A recent study confirmed UGT1A1*93 as a more robust marker for neutropenia than UGT1A1*2816.

The three ABC transporter SNPs in the final multivariable models are in noncoding regions, either intronic or upstream of their respective genes, and are not in high linkage disequilibrium with any coding SNPs. This was expected, considering the low number of high-frequency coding SNPs in these genes. Previous studies of the effects of nonsynonymous SNPs on the expression and activity of these ABC transporters have yielded mostly negative or inconclusive results40–42. Of the SNPs with P < 0.15 in the univariate analyses, only one (rs1128503) is in a coding region and one intronic SNP (rs2373586) is in high LD with a common missense SNP, rs2032582. rs1128503 and rs2032582 are both in ABCB1 and, together with rs1045642, make up the ABCB1*2 haplotype. This haplotype has been previously associated with early toxicity during irinotecan treatment26. However, these SNPs dropped out of significance during the selection of the multivariable models. These coding SNPs have been frequently studied together and individually for association with transporter activity and changes in exposure and response to numerous drugs, but with discordant results40.

In contrast to coding SNPs, due to lower selective pressures and more modest effects, variants in the regulatory regions of membrane transporter genes are more abundant and exist at higher allele frequencies43,44. Functional studies of variants in the proximal promoter44–47, untranslated48,49 and intronic50,51 regions of ABC and SLCO transporter genes have demonstrated changes in mRNA or protein expression levels and interindividual variation in pharmacokinetics52,53. In light of this, it is likely that noncoding genetic variation has a more widespread impact on transporter function. Our results highlight the importance of including noncoding SNPs in pharmacogenetic studies and the need to further characterize regulatory regions in pharmacogenes.

The significant variants identified in this study may contribute to irinotecan-induced neutropenia by altering expression of ABCB1 or ABCC1. ABCB1 encodes P-glycoprotein, which is involved in irinotecan and SN-38 biliary excretion54,55. An increase in ABCB1 expression could result in increased biliary secretion of SN-38 and a corresponding reduction in plasma SN-38 levels, which are inversely correlated with ANC nadir. ABCC1 encodes MRP1, a hepatic basolateral efflux transporter of SN-3856. Increased ABCC1 expression is hypothesized to result in increased systemic levels of SN-38, which is consistent with the decreased ANC nadir associated with this variant. It should be noted that ABCC1/MRP1 expression in the liver is relatively low57, so increased expression may have a significant impact on basolateral efflux of SN-38 back into the blood.

Bioinformatic analyses predicted 5′-flanking ABCC1 SNP rs6498588 to alter TF binding and rs4148330 (in LD with rs6498588) to have both enhancer activity in hepatic cells and effects on TF binding. A preliminary eQTL meta-analysis of human liver samples found rs6498588 to be significantly associated (meta-adjusted P = 0.05) with increased ABCC1 expression58. These data are consistent with the association of rs6498588 with increased SN-38 AUC, as higher ABCC1 expression would result in increased transport of SN-38 into the blood. However, our in vitro enhancer assays did not confirm these predictions for either rs6498588 or rs4148330. Enhancer assays conducted in additional hepatic cell lines or with different-sized genomic regions are warranted.

A combined allele analysis showed that a higher number of risk alleles from our models is correlated with irinotecan-induced toxicity. A significantly greater proportion of patients with grade 3–4 neutropenia carried at least four risk alleles for both phenotypes when compared to the group with no or less severe neutropenia. rs12720066 and UGT1A1*93 appear to be driving this effect, with the directions of their effects consistent with the toxicity in both pharmacokinetic and pharmacodynamic outcomes. While rs6498588 was only in the final SN-38 AUC model, a SNP in high LD (rs8050881) had a weaker univariate association with ANC nadir; the variant alleles of these SNPs are also associated with the toxicity in both outcomes. However, the contribution of rs17501331 is less clear; this variant was only associated with ANC nadir and not at all with SN-38 AUC, suggesting a mechanism that may affect neutrophil count independent of SN-38 exposure. Given the complexity of the many metabolizing enzymes and transporters acting on SN-38, further eQTL and functional characterization studies are needed to validate the effect of these SNPs before proposing a combined PK/PD model.

The addition of rs6498588 to our previously published model14 of log SN-38 AUC containing rs35605 (ABCC1), rs10276036 (ABCB1) and UGT1A1*93 improved the percent variation of SN-38 AUC explained by genetic determinants (adjusted R2 increased from 0.221 to 0.284, P = 0.009, ANOVA), with all four variants in the model remaining significant. When rs12720066 and rs17501331 were added to our previous model of log ANC nadir14 containing rs3765129 (ABCC1), rs2306283 (SLCO1B1), and UGT1A1*93, only rs17501331 had marginal significance (P = 0.05) and a smaller effect than the other variants in the initial model. The previously identified SNPs, especially UGT1A1*93, may explain more of the genetic variation in ANC nadir. Again, a better mechanistic understanding of this toxicity is needed before proposing a predictive multivariable model for irinotecan-induced neutropenia.

There are no known clinical associations of the variants identified in the current association studies. However, there is still enough compelling functional evidence to suggest that genetic variants in ABCB1 and ABCC1 are relevant to irinotecan toxicity. MRP1-overexpressing KB-3–1 cells showed increased SN-38 resistance compared to normal cells56, affirming that expression of the transporter does affect SN-38 transport. In ABCB1 knockout mice, irinotecan and SN-38 plasma levels were increased relative to the wildtype mice, consistent with a role for P-gp in biliary secretion of this drug/metabolite pair55. Cell lines overexpressing BCRP (encoded by ABCG2) are also resistant to irinotecan59 and SN-3860, and ABCG2 421C>A reduced gene expression and conferred irinotecan resistance in cancer cell lines61. In the current study, there was no association with any of the tested ABCG2 variants and SN-38 exposure or ANC nadir.

Other genes in the irinotecan pharmacokinetic pathway including ABCC2, SLCO1B1, CES1, CES2, CYP3A4 and additional variants in UGT1A1 and other UGT genes may also contribute to SN-38 exposure and neutropenia and warrant further study62–64. In contrast, genetic variation in genes responsible for irinotecan pharmacodynamics do not seem to play a role65. More comprehensive genotyping of pharmacokinetic genes has the potential to identify additional genetic variants more strongly associated with irinotecan exposure and toxicity. Discovery of additional genetic and non-genetic determinants of irinotecan-induced neutropenia will improve the predictive power of a clinical pharmacogenetic algorithm over using a single UGT1A1*28 test for drug selection and dosing. Replication and prospective studies in large independent cohorts as well as in other ethnic groups are necessary to validate and expand these findings.

A current gap in the field of pharmacogenetics is understanding the clinical value of variants. Our study used a pharmacokinetic/pharmacodynamic phenotyping approach to discover new variants associated with neutropenia during irinotecan treatment. Further investigation of these variants and others in ABC transporter genes may not only potentially contribute to the improvement of individualized treatment with irinotecan, but may also have broader implications for other substrates of ABC transporters.

Supplementary Material

Supplemental Data

Acknowledgments

This work was supported by NIH/NIGMS U01GM061390 (Pharmacogenetics of Membrane Transporters), NIH/NIGMS U01GM0061393 (PAAR-Pharmacogenomics of Anticancer Agents Research Group), NIH/NIGMS T32 GM007175, NIH/NIDDK R21DK081157, and NIH/NCI K07CA140390-01.

Footnotes

Conflict of Interest

Drs. Innocenti and Ratain are patent holders on the UGT1A1 testing for irinotecan neutropenia. Dr. Ratain also receives intermittent royalties on multiple patents related to irinotecan pharmacogenetics and is an inventor on a pending patent application for a genomic prescribing system. The remaining authors declare no conflicts of interest.

Supplementary information is available at The Pharmacogenomics Journal’s website.

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