ORIGINAL RESEARCH published: 30 June 2020 doi: 10.3389/fphar.2020.00973 ABCB1 Genetic Variants as Predictors of Irinotecan-Induced Severe Gastrointestinal Toxicity in Metastatic Colorectal Edited by: Cancer Patients Chonlaphat Sukasem, Mahidol University, Thailand Pau Riera 1,2,3†, Alı́cia Artigas-Baleri 1†, Juliana Salazar 4*, Ana Sebio 5, Anna C. Virgili 5, Marı́a Jesús Arranz 6 and David Páez 5,7 Reviewed by: Chalirmporn Atasilp, 1 Genetics Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 2 Faculty of Pharmacy and Food Sciences, Rangsit University, Thailand Universitat de Barcelona (UB), Barcelona, Spain, 3 Pharmacy Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Luis Andrés López-Fernández, Spain, 4 Translational Medical Oncology Laboratory, Institut de Recerca Biomèdica Sant Pau, (IIB-Sant Pau), Barcelona, Gregorio Marañón Hospital, Spain Spain, 5 Medical Oncology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 6 Research Laboratory, Jaw-Yuan Wang, Fundació Docència i Investigació Mútua Terrassa, Terrassa, Spain, 7 U705, ISCIII Center for Biomedical Research on Rare Kaohsiung Medical University, Taiwan Diseases (CIBERER), Barcelona, Spain *Correspondence: Juliana Salazar jsalazar@santpau.cat Irinotecan is widely used in the treatment of metastatic colorectal cancer (mCRC) despite † These authors have contributed its severe toxicities. Toxicity is often associated with the UGT1A1*28/*28 genotype. An equally to this work explanation for idiopathic toxicity beyond the UGT1A1 biomarker, however, remains a major concern for clinicians. One of the main irinotecan transporters is P-glycoprotein (P- Specialty section: This article was submitted to gp), which is a hepatic efflux pump encoded by ABCB1. P-gp is involved in the biliary Pharmacogenetics excretion of irinotecan and its active metabolite SN-38. We aimed to assess whether and Pharmacogenomics, functional variants in ABCB1 also contribute to identifying patients at risk of toxicity. A a section of the journal Frontiers in Pharmacology cohort of 308 mCRC patients treated with irinotecan-based regimens were genotyped for Received: 07 April 2020 polymorphisms in ABCB1 (rs1128503, rs2032582, and rs1045642). The effect of these Accepted: 15 June 2020 variants and their haplotypes on irinotecan-induced severe toxicity (diarrhea, neutropenia, Published: 30 June 2020 asthenia, nausea, and mucositis) was assessed. After adjusting for the relevant clinical and Citation: Riera P, Artigas-Baleri A, pathological parameters in the multivariate analysis, we found rs1128503 was significantly Salazar J, Sebio A, Virgili AC, associated with severe diarrhea and mucositis (P=0.014 and P=0.002, respectively). Arranz MJ and Páez D (2020) Additionally, rs2032582 was associated with severe mucositis (P<0.001). Our results ABCB1 Genetic Variants as Predictors of Irinotecan-Induced show that rs1128503 genotyping could help to predict severe gastrointestinal toxicity Severe Gastrointestinal Toxicity in induced by irinotecan. Metastatic Colorectal Cancer Patients. Front. Pharmacol. 11:973. Keywords: ABCB1, P-glycoprotein, irinotecan (CPT-11), gastrointestinal toxicity, diarrhea, mucositis, biomarker, doi: 10.3389/fphar.2020.00973 colorectal cancer Frontiers in Pharmacology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity INTRODUCTION 125–350 mg/m2). We excluded those patients without available germline DNA or without all the toxicities properly collected. Irinotecan (CPT-11) is an antitumor agent that is broadly used in The toxicities assessed were diarrhea, neutropenia, asthenia, nausea, metastatic colorectal cancer (mCRC) patients and normally co- and mucositis. For each side-effect, we recorded the highest grade of administered with infusional 5-fluorouracil/leucovorin (FOLFIRI toxicity experienced by each patient. Toxicities were graded (1 to 4) regimen). Its use is hampered by toxicities such as severe diarrhea on the basis of the Common Terminology Criteria for Adverse and neutropenia in more than one-third of the patients (Fuchs Events v5.0 (CTCAE) of the National Cancer Institute. Grade 3 to 4 et al., 2003). toxicities were considered severe toxicities. The study was approved In the liver, CPT-11 is converted to 7-ethyl-10-hydroxy- by the Institutional Ethics Committee at HSCSP and written camptothecin (SN-38), which is responsible for both the efficacy informed consent was obtained from all participants. and the toxicity of the drug. SN-38 is mainly eliminated through glucuronidation by the uridine 5′-diphospho-(UDP)- Genotyping glucuronosyltransferase (UGT) isoform 1A1, which is encoded by Germline DNA was extracted from blood cells by the salting-out the UGT1A1 gene. The presence of seven TA repeats (UGT1A1*28 procedure (Miller et al., 1988). Three functional ABCB1 gene allele) in the promoter region of this gene reduces enzymatic activity polymorphisms, rs1128503 (1236 C>T), rs2032582 (2677 G>T/ (Bosma et al., 1995) and patients homozygous for this variant have a A), and rs1045642 (3435 C>T), were genotyped using TaqMan higher risk of irinotecan-induced toxicity (Innocenti et al., 2004; PCR assays (Applied Biosystems, Foster City, CA, USA). Due to Marcuello et al., 2004; Rouits et al., 2004; Riera et al., 2018). the rs2032582 triallelic condition, two probes (C_11711720C_30 However, UGT1A1 genetic status does not always explain severe and C_11711720D_40) were needed to genotype this variant. toxicity, suggesting that other mechanisms contribute to the The other probes used were C_7586662_10 for rs11128503, and appearance of side effects. C_7586657_20 for rs1045642. Genotyping was performed on a An ATP-powered pump encoded by the ABCB1 gene, P- 7500 Real Time PCR System (Applied Biosystems, Foster City, glycoprotein (P-gp), participates in the biliary excretion of CPT- CA, USA). 11 and SN-38. The enhancement of P-gp expression increases biliary secretion of SN-38 and reduces its plasma concentrations, Statistics conferring a higher risk of intestinal toxicity but a lower risk of Allele frequencies for each single nucleotide polymorphism (SNP) neutropenia (Iyer et al., 2002; Mathijssen et al., 2003; Han et al., were checked by direct counting and compared to published data 2009; Filipski et al., 2014; Li et al., 2018). The most well-known (1000 Genomes Project Consortium et al., 2015). Genotype data ABCB1 variants, rs1128503 (1236 C>T), rs2032582 (2677 G>T/A), were tested for Hardy-Weinberg equilibrium using chi-squared and rs1045642 (3435 C>T), modulate P-gp expression (Hoffmeyer tests. The triallelic condition of the rs2032582 variant results in et al., 2000; Haenisch et al., 2007), CPT-11 and SN-38 plasma six possible genotypes (GG, GT, GA, TT, TA, and AA). To concentrations, and renal clearance (Mathijssen et al., 2003; Sai simplify the statistical analyses, we combined patients with GT or et al., 2003). However, the influence of these variants on irinotecan- GA genotypes, and also patients with TT, TA, or AA genotypes. induced severe toxicity remains inconclusive (Han et al., 2007; For haplotype analysis, GA, TA, and AA patients were excluded, Innocenti et al., 2009; Glimelius et al., 2011; Teft et al., 2015; Li et al., as PLINK removes triallelic SNP genotype calls. We performed 2018; Salvador-Martı́n et al., 2018). phased haplotype analyses (using the E-M algorithm) including Based on the relevance of P-gp on irinotecan pharmacokinetics, the three ABCB1 variants or using the window option for diplotype we conducted a study in a cohort of mCRC patients treated with an analyses. Associations between toxicities and genetic variants/ irinotecan-containing therapy in whom the UGT1A1 gene had haplotypes were measured using chi-squared tests. Multivariate previously been analyzed. The aim was to elucidate whether the analyses were carried out, including sex, age, chemotherapy aforementioned ABCB1 variants could also help to identify patients treatment, line of chemotherapy, and UGT1A1 and ABCB1 who develop severe irinotecan-induced toxicity. genotypes. P-values <0.05 were considered statistically significant. Bonferroni correction for multiple comparisons was applied (P- values <2.4·10−4). All statistical analyses were conducted using SPSS MATERIAL AND METHODS (v25.0, IBM) and PLINK (v1.07, Shaun Purcell). Study Population We retrospectively included patients from Hospital de la Santa RESULTS Creu i Sant Pau (HSCSP, Barcelona, Spain) who were diagnosed between 2001 and 2016 with mCRC and treated with an Patient Population irinotecan-containing regimen. All patients had an Eastern We studied a total of 308 patients. Most patients (n=283) were Cooperative Oncology Group (ECOG) performance status ≤2 treated with irinotecan in combination with 5-fluorouracil and and age ≥ 18. Irinotecan-containing regimens were biweekly leucovorin (FOLFIRI). A total of 176 patients had been enrolled FOLFIRI (irinotecan 180 mg/m2), irinotecan in monotherapy in clinical trials (Marcuello et al., 2011; Pá ez et al., 2019). The every 3 weeks (350 mg/m2, except for one patient with the presence of the UGT1A1*28, UGT1A1*37, and UGT1A1*36 UGT1A1*28/*37 genotype who received a reduced dose of 150 alleles was determined in a previous study (Riera et al., 2018). mg/m2), and other regimens containing irinotecan (irinotecan Table 1 shows patients’ clinical characteristics along with Frontiers in Pharmacology | www.frontiersin.org 2 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity UGT1A1 genotype status. Supplementary Table 1 shows toxicity induced side effects. In the multivariate analysis, rs1128503 was grades according to CTCAE v5.0. significantly associated with severe diarrhea and mucositis (P=0.014 and P=0.002, respectively). Patients harboring the CC genotype for Genetic Studies rs1128503 presented a higher prevalence of severe diarrhea and Allelic frequencies for all polymorphisms were within the mucositis than those with CT and TT genotypes (Figure 1). In probability limits of Hardy–Weinberg equilibrium (P>0.05), addition, rs2032582 was associated with severe mucositis and their minor allele frequencies were in accordance with (P=0.0001). This association retained its significance after European population frequencies reported in the 1000 Bonferroni correction. Table 2 shows the genotyping distributions Genomes Project (1000 Genomes Project Consortium et al., and associations between the three polymorphisms and the toxicities 2015). Genotyping was successful in 307 patients (99.7%). assessed. When analyzing gene interactions, we found no significant interactions between the UGT1A1 and the ABCB1 variants tested. Associations Between ABCB1 Variants and Severe Side Effects Associations Between ABCB1 Haplotypes and In the univariate analysis, we found no significant associations between the tested ABCB1 variants and the presence of irinotecan- Severe Side Effects In the univariate analysis, we found that the diplotypes rs1045642T-rs1128503C (frequency=10.5%) and rs1128503C- rs2032582T (frequency=1.9%) were associated with a higher TABLE 1 | Baseline patient characteristics (n=308). risk of diarrhea (P=0.034 and P=0.002, respectively). The n % rs1128503C-rs2032582T diplotype was also associated with a higher risk of mucositis (P=3.8·10−8), asthenia (P=0.043), and Sex Male 194 63.0% nausea (P=0.034). The association with mucositis remained Female 114 37.0% statistically significant after applying Bonferroni test. Age In the multivariate analysis, the associations of both diplotypes Mean 63 with diarrhea remained significant (P=0.035 for rs1045642T- Range 24–83 rs1128503C and P=0.005 for rs1128503C-rs2032582T). The Primary tumor site Right colon 76 24.7% associations of the diplotype rs1128503C-rs2032582T with Left colon 232 75.3% mucositis and nausea also retained their significance after Chemotherapy treatment multivariate adjustments (P=0.00018 and P=0.049, respectively), FOLFIRI 283 91.9% but only the association with mucositis retained its significance after Irinotecan monotherapy 13 4.2% Other irinotecan combinations 12 3.9% Bonferroni correction. No other associations between ABCB1 Line of chemotherapy diplotypes and the occurrence of severe side effects were found. First line 244 79.2% These results are shown in the Supplementary Table 2. ≥ Second line 64 20.8% The haplotype analyses considering the three ABCB1 gene UGT1A1 genotype variants (rs1128503-rs2032582-rs1045642) identified seven of UGT1A1 *1/*1 136 44.2% UGT1A1 *1/*28 143 46.4% the eight possible haplotypes in our cohort of patients. The UGT1A1 *28/*28 28 9.1% most frequent haplotypes were CGC and TTT, with a prevalence UGT1A1 *28/*37 1 0.3% of 47.5% (n=282) and 37.4% (n=222), respectively (Table 3). In FOLFIRI, combination of irinotecan, 5-fluorouracil, and leucovorin, UGT1A1, uridine 5′- contrast, the haplotype CTC was rare (0.3%) and the TGT diphospho-(UDP)-glucuronosyltransferase (UGT) isoform 1A1 haplotype was not harbored by any patient. A B FIGURE 1 | Association between rs1128503 genotype and irinotecan-induced gastrointestinal severe toxicities [(A) diarrhea; (B) mucositis)]. Frontiers in Pharmacology | www.frontiersin.org 3 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity TABLE 2 | Univariate and multivariate associations between ABCB1 genotypes and grade 3–4 toxicities. Genotype n (%) Diarrhea, n (%) Neutropenia, n (%) Asthenia, n (%) Nausea, n (%) Mucositis, n (%) rs1128503 (1236 C>T) CC 107 (34.9%) 22 (20.6%) 29 (27.1%) 24 (22.4%) 10 (9.4%) 5 (4.7%) CT 145 (47.2%) 24 (16.6%) 27 (18.6%) 29 (20.0%) 11 (7.6%) 4 (2.8%) TT 55 (17.9%) 9 (16.4 %) 11 (20.0%) 11 (20.0%) 6 (10.9%) 0 (0%) P-value (univariate analysis) P = 0.676 P = 0.256 P = 0.883 P = 0.737 P = 0.245 P-value (multivariate analysis) P = 0.014 P = 0.990 P = 0.236 P = 0.615 P = 0.002 rs2032582 (2677 G>T/A) GG 102 (33.2%) 18 (17.7%) 28 (27.5%) 20 (19.6%) 8 (7.8%) 2 (2.0%) GT 144 (46.9%) GA 7 (2.3%) GT+GA 26 (17.2%) 28 (18.5%) 33 (21.9%) 14 (9.3%) 5 (3.3%) TT 51 (16.6%) TA 3 (1.0%) TT+TA 11 (20.4%) 11 (20.4%) 11 (20.4%) 5 (9.3%) 2 (3.7%) P-value (univariate analysis) P = 0.871 P = 0.233 P = 0.907 P = 0.917 P = 0.768 P-value (multivariate analysis) P = 0.095 P = 0.213 P = 0.586 P = 0.713 P = 0.0001a rs1045642 (3435 C>T) CC 90 (29.3%) 15 (16.7%) 24 (26.7%) 16 (17.8%) 6 (6.7%) 3 (3.3%) CT 149 (48.5%) 25 (16.8%) 27 (18.1%) 34 (22.8%) 15 (10.1%) 6 (4.0%) TT 68 (22.2%) 15 (22.1%) 16 (23.5%) 14 (20.6%) 6 (8.8%) 0 (0%) P-value (univariate analysis) P = 0.600 P = 0.279 P = 0.648 P = 0.667 P = 0.255 P-value (multivariate analysis) P = 0.197 P = 0.207 P = 0.240 P = 0.659 P = 0.352 The bold values indicate the statistically significant P-values (P < 0.05). a Significant after Bonferroni correction (P < 2.4·10−4). ABCB1, ATP binding cassette subfamily B member 1. In the univariate analysis, the CTT haplotype (frequency= of evidence shows that variants in CPT-11 transporter genes could 1.5%) was significantly associated with a higher risk of severe be helpful to identify patients at risk of severe side effects (Innocenti diarrhea, mucositis, asthenia, and nausea (P=0.005, P=8.8·10−6, et al., 2009; Chen et al., 2015; Teft et al., 2015). ABCB1 gene is one P=0.015, and P=0.014, respectively). These associations of the most promising candidates, considering the relevance of the retained their significance after multivariate adjustments P-gp on the biliary secretion of irinotecan and its metabolites from (P=0.012, P=0.002, P=0.047, and P=0.029, respectively) (see the liver (Iyer et al., 2002). Great attention has been paid to Table 3). Only the association with mucositis in the univariate rs1128503 (1236 C>T), rs2032582 (2677 G>T/A), and rs1045642 analysis remained statistically significant after applying the (3435 C>T), variants found to be in strong linkage disequilibrium Bonferroni test. and associated with drug exposure (Mathijssen et al., 2003; Han et al., 2007; Li et al., 2018). Rhodes et al. analyzed 11 polymorphisms in 54 patients with DISCUSSION metastatic colorectal cancer, and reported significant associations with grade 3–4 toxicity and grade 3–4 neutropenia when combining This study shows the ABCB1 rs1128503 variant is a promising ABCB1, C1236T, and SLCO1B1 T521C polymorphisms (Rhodes predictor of irinotecan-induced severe gastrointestinal toxicity, et al., 2007). More recently, Salvador-Martı́n et al. analyzed the in particular diarrhea and mucositis. This result is reinforced by association between variants in ABC efflux transporter genes and the significant associations found between the diplotypes and irinotecan adverse effects, and found that the ABCB1 variants were haplotypes containing the rs1128503-C allele and the referred significantly associated with overall toxicity (P<0.01) and with toxicities. The rs2032582 variant also appears to be associated higher hematological toxicity including, but not limited to, with severe mucositis. neutropenia (P<0.01) (Salvador-Martín et al., 2018). However, Irinotecan-induced toxicities, the most common being other studies conducted in mCRC patients treated with irinotecan neutropenia and diarrhea, usually lead to dose reduction or in whom ABCB1 variants were analyzed did not show significant treatment withdrawal (Douillard et al., 2000). The Food and associations with severe neutropenia (De Mattia et al., 2013; Chen Drug Administration (FDA) drug label for irinotecan includes et al., 2015; Teft et al., 2015; Yan et al., 2016). Our findings support therapeutic recommendations for UGT1A1*28 homozygous these negative results as we found no associations with severe patients to reduce the risk of developing neutropenia [Irinotecan neutropenia in the 307 patients analyzed. These data, together (CAMPTOSAR) drug label. Food and Drug Administration. with the significant association obtained with the UGT1A1*28 (Accession date: 24/02/2020)]. However, no recommendations marker (P = 0.037) in a previous study by our group (Riera et al., are given to reduce the likelihood of irinotecan-induced severe 2018), lead us to infer that UGT1A1 is a more powerful predictor of gastrointestinal toxicity. The variability observed in the severe neutropenia than ABCB1. development of toxicity in clinical practice suggests that other Few studies have analyzed ABCB1 variants as determinants of polymorphisms or haplotypes could be involved. A growing body gastrointestinal toxicity in mCRC patients treated with Frontiers in Pharmacology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity TABLE 3 | Univariate and multivariate associations between ABCB1 haplotypes and grade 3–4 toxicities. Haplotype (rs1128503-rs2032582-rs1045642)* TTT (n=222) TTC (n=13) CTT (n=9) TGC (n=16) CGT (n=50) CGC (n=282) Diarrhoea Unaffected (n=485) 185 (38.1%) 10 (2.1%) 4 (0.8%) 15 (3.1%) 38 (7.8%) 233 (48.0%) Affected (n=107) 37 (34.6%) 3 (2.8%) 5 (4.7%) 1 (0.9%) 12 (11.2%) 49 (45.8%) P-value (univariate analysis) 0.511 0.703 0.005 0.213 0.256 0.680 P-value (multivariate analysis) 0.459 0.888 0.012 0.193 0.306 0.791 Neutropenia Unaffected (n=458) 179 (39.1%) 10 (2.2%) 5 (1.1%) 13 (2.8%) 38 (8.3%) 213 (46.5%) Affected (n=134) 43 (32.1%) 3 (2.2%) 4 (3.0%) 3 (2.2%) 12 (9.0%) 69 (51.5%) P-value (univariate analysis) 0.151 0.963 0.140 0.708 0.812 0.303 P-value (multivariate analysis) 0.122 0.631 0.234 0.815 0.361 0.408 Asthenia Unaffected (n=468) 176 (37.6%) 12 (2.6%) 4 (0.9%) 13 (2.8%) 40 (8.5%) 223 (47.6%) Affected (n=124) 46 (37.1%) 1 (0.8%) 5 (4.0%) 3 (2.4%) 10 (8.1%) 59 (47.6%) P-value (univariate analysis) 0.935 0.246 0.015 0.829 0.901 0.984 P-value (multivariate analysis) 0.975 0.203 0.047 0.979 0.765 0.777 Nausea Unaffected (n=538) 201 (37.4%) 13 (2.4%) 6 (1.1%) 14 (2.6%) 47 (8.7%) 257 (47.8%) Affected (n=54) 21 (38.9%) 0 (0%) 3 (5.6%) 2 (3.7%) 3 (5.6%) 25 (46.3%) P-value (univariate analysis) 0.812 0.263 0.014 0.633 0.447 0.832 P-value (multivariate analysis) 0.799 0.708 0.029 0.753 0.427 0.866 Mucositis Unaffected (n=579) 219 (37.8%) 13 (2.2%) 7 (1.2%) 16 (2.8%) 50 (8.6%) 274 (47.3%) Affected (n=13) 3 (23.1%) 0 (0%) 2 (15.4%) 0 (0%) 0 (0%) 8 (61.5%) P-value (univariate analysis) 0.214 0.906 8.8·10−6a 0.541 0.302 0.353 P-value (multivariate analysis) 0.251 0.921 0.002 0.998 0.458 0.733 *The haplotype CTC was present in two patients. Due to its low frequency, it was excluded from the statistical analysis. The haplotype TGT was not harbored by any patient. a Significant after Bonferroni correction (P < 2.4·10-4). The bold values indicate the statistically significant P-values (P < 0.05). ABCB1, ATP binding cassette subfamily B member 1. irinotecan, and most of them produced negative results (De similar side-effect profile of 5-fluorouracil and irinotecan. Mattia et al., 2013; Chen et al., 2015; Teft et al., 2015; Yan et al., However, the ABCB1 transporter has not been found to be 2016; Salvador-Martı́n et al., 2018). Nevertheless, in a cohort of involved in the pharmacokinetics of 5-fluorouracil, suggesting 56 mCRC patients receiving an irinotecan-based treatment, that the associations observed are likely due to irinotecan effects. Cortejoso et al. found that patients with the CC genotype for A third limitation is that we did not record whether toxicity the rs1045642 variant had a higher probability of diarrhea than appeared in the first or later cycles or whether diarrhea was early- patients with CT and TT genotypes (P=0.039) (Cortejoso, et al., onset or delayed. Neither did we record the prophylactic anti- 2013). In line with this study, and after adjusting for diarrheal agents received by each patient. Taken together, these clinicopathological covariates and the UGT1A1*28 genotype in data would have been useful to interpret the results. Finally, the a larger sample, we found that patients with the CC genotype for associations found in this study should be considered the ABCB1 rs1128503 variant were at highest risk of diarrhea exploratory because only the results with a P<0.001 survived (P=0.014) and mucositis (P=0.002). Furthermore, diplotypes and Bonferroni correction for multiple comparisons. We note, haplotypes including the rs1128503-C allele were significantly however, that the Bonferroni correction was too conservative associated with severe gastrointestinal toxicity, suggesting this as the ABCB1 variants were in linkage disequilibrium and the allele contributes to the risk of toxicity. Of note, Mathijssen et al. procedure ignored the dependencies between these variants. This reported that patients homozygous for the rs1128503-T allele limitation could be overcome by validating our findings in a showed greater exposure to both irinotecan (P=0.038) and SN-38 similar and independent cohort of patients, and also by (P=0.031) than the other patients in the trial (Mathijssen et al., performing functional studies. 2003). They also postulated that this synonymous variant could In conclusion, our findings in a large and homogeneous cohort alter RNA stability and affect the expression of ABCB1. Thus, in of mCRC patients show that rs1128503 and the diplotypes/ keeping with our findings, we speculate that patients carrying the haplotypes containing the C allele of this variant could be useful TT genotype may present less biliary excretion and a lower risk predictors of irinotecan-induced severe gastrointestinal toxicity. of gastrointestinal toxicity. Further studies are needed to increase evidence regarding the utility In an attempt to improve the pharmacogenetic prediction of of the rs1128503 variant in clinical practice. irinotecan-induced severe toxicity, we focused our study on the three most informative ABCB1 variants. Although this approach allowed us to achieve a higher statistical power, it has the DATA AVAILABILITY STATEMENT limitation of losing the genetic effect of other variants located in the ABCB1 gene or in other hepatic efflux transporters, such as The raw data supporting the conclusions of this article will be ABCC2 or ABCG2. A second limitation of our study could be the made available by the authors, without undue reservation. Frontiers in Pharmacology | www.frontiersin.org 5 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity ETHICS STATEMENT authors contributed to the article and approved the submitted version. The studies involving human participants were reviewed and approved by Institutional Ethics Committee at Hospital de la Santa Creu i Sant Pau. The patients/participants provided their ACKNOWLEDGMENTS written informed consent to participate in this study. This work was supported by the Instituto de Salud Carlos III (CM18/00207 for PR). AUTHOR CONTRIBUTIONS PR, AA-B, JS, and DP contributed to the conception and SUPPLEMENTARY MATERIAL design of the study. PR, AA-B, and JS performed genotyping and collected data. AS and AV collected data. PR, AA-B, JS, The Supplementary Material for this article can be found online MA, and DP analyzed the data and performed statistical at: https://www.frontiersin.org/articles/10.3389/fphar.2020.00973/ analyses. PR, AA-B, JS, and DP wrote the manuscript. All full#supplementary-material REFERENCES patients with advanced non-small cell lung cancer. Lung Cancer 63 (1), 115– 120. doi: 10.1016/j.lungcan.2007.12.003 1000 Genomes Project Consortium, R. A., Auton, A., Brooks, L. D., Durbin, R. M., Hoffmeyer, S., Burk, O., von Richter, O., Arnold, H. P., Brockmöller, J., Johne, A., Garrison, E. P., Kang, H. M., et al. (2015). A global reference for human genetic et al. (2000). Functional polymorphisms of the human multidrug-resistance variation. Nature 526 (7571), 68–74. doi: 10.1038/nature15393 gene: Multiple sequence variations and correlation of one allele with P- Bosma, P. J., Chowdhury, J. R., Bakker, C., Gantla, S., de Boer, A., Oostra, B. A., glycoprotein expression and activity in vivo. Proc. Natl. Acad. Sci. 97 (7), et al. (1995). The genetic basis of the reduced expression of bilirubin UDP- 3473–3478. doi: 10.1073/pnas.050585397 glucuronosyltransferase 1 in Gilbert’s syndrome. New Engl. J. Med. 333 (18), Innocenti, F., Undevia, S. D., Iyer, L., Chen, P. X., Das, S., Kocherginsky, M., et al. 1171–1175. doi: 10.1056/NEJM199511023331802 (2004). Genetic variants in the UDP-glucuronosyltransferase 1A1 gene predict Chen, S., Villeneuve, L., Jonker, D., Couture, F., Laverdière, I., Cecchin, E., et al. the risk of severe neutropenia of irinotecan. J. Clin. Oncol. 22 (8), 1382–1388. (2015). ABCC5 and ABCG1 polymorphisms predict irinotecan-induced severe doi: 10.1200/JCO.2004.07.173 toxicity in metastatic colorectal cancer patients. Pharmacogenet. Genomics 25 Innocenti, F., Kroetz, D. L., Schuetz, E., Dolan, M. E., Ramı́rez, J., Relling, M., et al. (12), 573–583. doi: 10.1097/FPC.0000000000000168 (2009). Comprehensive Pharmacogenetic Analysis of Irinotecan Neutropenia Cortejoso, L., Garcı́a, M. I., Garcı́a-Alfonso, P., Gonzá lez-Haba, E., Escolar, F., and Pharmacokinetics. J. Clin. Oncol. 27 (16), 2604–2614. doi: 10.1200/ Sanjurjo, M., et al. (2013). Differential toxicity biomarkers for irinotecan- and JCO.2008.20.6300 oxaliplatin-containing chemotherapy in colorectal cancer. Cancer Chemother. Irinotecan (CAMPTOSAR) drug label. Food and Drug Administration. Pharmacol. 71 (6), 1463–1472. doi: 10.1007/s00280-013-2145-6 [Accession date: 02/24/2020]. De Mattia, E., Toffoli, G., Polesel, J., D’Andrea, M., Corona, G., Zagonel, V., et al. Iyer, L., Ramı́rez, J., Shepard, D. R., Bingham, C. M., Hossfeld, D.-K., Ratain, M. J., (2013). Pharmacogenetics of ABC and SLC transporters in metastatic colorectal et al. (2002). Biliary transport of irinotecan and metabolites in normal and P- cancer patients receiving first-line FOLFIRI treatment. Pharmacogenet. Genomics glycoprotein-deficient mice. Cancer Chemother Pharmacol. 49 (4), 336–341. 23 (10), 549–557. doi: 10.1097/FPC.0b013e328364b6cf doi: 10.1007/s00280-001-0420-4 Douillard, J. Y., Cunningham, D., Roth, A. D., Navarro, M., James, R. D., Karasek, Li, M., Seiser, E. L., Baldwin, R. M., Ramirez, J., Ratain, M. J., Innocenti, F., et al. P., et al. (2000). Irinotecan combined with fluorouracil compared with (2018). ABC transporter polymorphisms are associated with irinotecan fluorouracil alone as first-line treatment for metastatic colorectal cancer: a pharmacokinetics and neutropenia. Pharmacogenom. J. 18 (1), 35–42. multicentre randomised trial. Lancet 355 (9209), 1041–1047. doi: 10.1016/ doi: 10.1038/tpj.2016.75 s0140-6736(00)02034-1 Marcuello, E., Alté s, A., Menoyo, A., Del Rio, E., Gó mez-Pardo, M., and Baiget, M. Filipski, E., Berland, E., Ozturk, N., Guettier, C., van der Horst, G. T. J., Lé vi, F., (2004). UGT1A1 gene variations and irinotecan treatment in patients with et al. (2014). Optimization of irinotecan chronotherapy with P-glycoprotein metastatic colorectal cancer. Br. J. Cancer 91 (4), 678–682. doi: 10.1038/ inhibition. Toxicol. Appl. Pharmacol. 274 (3), 471–479. doi: 10.1016/ sj.bjc.6602042 j.taap.2013.12.018 Marcuello, E., Pá ez, D., Paré , L., Salazar, J., Sebio, A., del Rio, E., et al. (2011). A Fuchs, C. S., Moore, M. R., Harker, G., Villa, L., Rinaldi, D., and Hecht, J. R. (2003). genotype-directed phase I-IV dose-finding study of irinotecan in combination Phase III comparison of two irinotecan dosing regimens in second-line therapy with fluorouracil/leucovorin as first-line treatment in advanced colorectal of metastatic colorectal cancer. J. Clin. Oncol. 21 (5), 807–814. doi: 10.1200/ cancer. Br. J. Cancer 105 (1), 53–57. doi: 10.1038/bjc.2011.206 JCO.2003.08.058 Mathijssen, R. H. J., Marsh, S., Karlsson, M. O., Xie, R., Baker, S. D., Verweij, J., et al. Glimelius, B., Garmo, H., Berglund, A., Fredriksson, L. A., Berglund, M., Kohnke, H., (2003). Irinotecan pathway genotype analysis to predict pharmacokinetics. Clin. et al. (2011). Prediction of irinotecan and 5-fluorouracil toxicity and response in Cancer Res. 9 (9), 3246–3253. patients with advanced colorectal cancer. Pharmacogenom. J. 11 (1), 61–71. Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988). A simple salting out doi: 10.1038/tpj.2010.10 procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. Haenisch, S., Zimmermann, U., Dazert, E., Wruck, C. J., Dazert, P., Siegmund, S., 16 (3), 1215. doi: 10.1093/nar/16.3.1215 et al. (2007). Influence of polymorphisms of ABCB1 and ABCC2 on mRNA Pá ez, D., Tobeña, M., Ferná ndez-Plana, J., Sebio, A., Virgili, A. C., Cirera, L., and protein expression in normal and cancerous kidney cortex. et al. (2019). Pharmacogenetic clinical randomised phase II trial to evaluate Pharmacogenom. J. 7 (1), 56–65. doi: 10.1038/sj.tpj.6500403 the efficacy and safety of FOLFIRI with high-dose irinotecan (HD- Han, J.-Y., Lim, H.-S., Yoo, Y.-K., Shin, E. S., Park, Y. H., Lee, S. Y., et al. (2007). FOLFIRI) in metastatic colorectal cancer patients according to their Associations of ABCB1, ABCC2, and ABCG2 polymorphisms with irinotecan- UGT1A 1 genotype. Br. J. Cancer 120 (2), 190–195. doi: 10.1038/s41416- pharmacokinetics and clinical outcome in patients with advanced non-small 018-0348-7 cell lung cancer. Cancer 110 (1), 138–147. doi: 10.1002/cncr.22760 Rhodes, K. E., Zhang, W., Yang, D., Press, O. A., Gordon, M., Vallböhmer, D., Han, J.-Y., Lim, H.-S., Park, Y. H., Lee, S. Y., and Lee, J. S. (2009). Integrated et al. (2007). ABCB1, SLCO1B1 and UGT1A1 gene polymorphisms are pharmacogenetic prediction of irinotecan pharmacokinetics and toxicity in associated with toxicity in metastatic colorectal cancer patients treated with Frontiers in Pharmacology | www.frontiersin.org 6 June 2020 | Volume 11 | Article 973 Riera et al. ABCB1 Variants and Irinotecan Gastrointestinal Toxicity first-line irinotecan. Drug Metab. Lett. 1 (1), 23–30. doi: 10.2174/ Yan, L., Wang, X., Wei, L., Nie, Y., Liu, J., and Zhang, L. (2016). Effects of UGT1A1*6, 187231207779814328 UGT1A1*28, and ABCB1-3435C