Effect of drug metabolizing enzymes and transporters in Thai colorectal cancer patients treated with irinotecan-based chemotherapy

Genetic polymorphisms in drug metabolizing enzymes and drug transporters may affect irinotecan toxicity. Although genetic polymorphisms have been shown to influence the irinotecan toxicity, data are limited in Thai population. Thus, the aim of this study was to assess the allele and genotype frequencies and the relationship between CYP3A4/5, DPYD, UGT1A1, ABCB1, and ABCC2 genetic variations and irinotecan-induced toxicity in Thai colorectal cancer patients. One hundred and thirty-two patients were genotyped, and the effect of genetic variations on irinotecan-induced toxicity was assessed in 66 patients who received irinotecan-based chemotherapy. Allele frequencies of ABCB1 c.1236C > T, ABCB1 c.3435C > T, ABCC2 c.3972C > T, ABCG2 c.421C > A, CYP3A4*1B, CYP3A4*18, CYP3A5*3, DPYD*5, UGT1A1*28, and UGT1A1*6 were 0.67, 0.43, 0.23, 0.27, 0.01, 0.02, 0.64, 0.19, 0.16, and 0.09, respectively. DPYD*2A and DPYD c.1774C > T variants were not detected in our study population. The ABCC2 c.3972C > T was significantly associated with grade 1–4 neutropenia (P < 0.012) at the first cycle. Patients carrying both UGT1A1*28 and *6 were significantly associated with severe neutropenia at the first (P < 0.001) and second (P = 0.017) cycles. In addition, patients carrying UG1A1*28 and *6 had significantly lower absolute neutrophil count (ANC) nadir at first (P < 0.001) and second (P = 0.001) cycles. This finding suggests that UGT1A1*28, *6, and ABCC2 c.3972C > T might be an important predictor for irinotecan-induced severe neutropenia.

Toxicity criteria. Toxicity was assessed at first and second cycles of treatment according to National Cancer Institute Common Toxicity Criteria for Adverse Events, version 5.0. Grade 3-4 toxicity was considered as severe toxicity.
Statistical analysis. Deviation from Hardy-Weinberg equilibrium was assessed using Fisher's exact and chi-square test. Allele and genotype frequencies were determined by direct counting. Comparisons of allele and genotype frequencies and grades of toxicity were performed using the χ 2 test. Mann-Whitney U test was performed according to difference of genetic groups and nonparametric data [absolute neutrophil count (ANC) nadir and ANC ratio]. Logistic regression analysis was performed to assess univariate and multivariate relationships genetic polymorphisms, and other parameters. All statistics were calculated using SPSS version 18 (SPSS Inc., Chicago, IL, USA) and differences were significant when P values were < 0.05.

Results
Clinical characteristics and genotyping data. Sixty-six patients with metastatic colorectal cancer receiving an irinotecan-based regimen were enrolled for association analysis. Their clinical characteristics are summarized in www.nature.com/scientificreports/ performance status of zero. The most common site of disease was the rectum. The liver was the dominant site for metastases. There were no statistically significant differences between clinical characteristics and hematological toxicity including neutropenia, leucopenia, thrombocytopenia, and anemia (data not shown).

First cycle Second cycle
Grade 1-4 a n (%) P

Discussion
In this study, the association between irinotecan-induced toxicity and pharmacogenetics of drug metabolizing enzymes and drug transporters was investigated. Our results showed that combined analysis of UGT1A1*28 and *6 polymorphisms and ABCC2 c.3972C > T were closely related with neutropenia toxicity in Thai colorectal cancer patients. The UGT1A1*28 allele is the most important risk factor for severe neutropenia or diarrhea. In 2005, the U.S. Food and Drug Administration (FDA) informed that patients with homozygous UGT1A1*28 are at increased Table 4. Multivariate logistic regression analysis to analyze the factors affecting neutropenia at first and second cycles. Exp exponential, 95% CI 95% confidence interval. † p value < 0.05 was considered statistically significant. a Grade 1-4 was considered as toxicity. b Grade 3-4 was considered as severe toxicity.  www.nature.com/scientificreports/ risk of severe neutropenia following initiation of irinotecan treatment 20 . Several studies have investigated the relationship of UGT1A1*28 and severe neutropenia and diarrhea during irinotecan treatment [21][22][23] . Wang et al. 24 reported a significantly high risk for grade 3-4 leukopenia and neutropenia in patients carrying heterozygous UGT1A1*28 compared to homozygous wild type patients. Similarly, Rouits et al. 25 reported that patients carrying the homozygous or heterozygous UGT1A1*28 had significantly higher risk of neutropenia than those with UGT1A1*1. In this study, although there were no significant differences between grade 3-4 neutropenia and patients with UGT1A1*28, the incidence of severe neutropenia in patients with hetero-and homozygous *28 was higher than patients with homozygous wild type at the first cycle (OR 2.7, 95% CI 0.8-8.8; P = 0.087). At the second cycle, patient carried UGT1A1*28 was a significantly higher risk of neutropenia than patients with homozygous wild type (OR 3.1, 95% CI 1.2-7.97; P = 0.017). A multivariate analysis was performed to analyze the influence of UGT1A1*28. This result shown that patients with UGT1A1*28 was at significant increased risk for grade 1-4 neutropenia compared with wild type patients (P = 0.011) in second cycle. Using ANC ratio, patient carried heterozygous and homozygous UGT1A1*28 had lower ANC nadir and ANC ratio than wild type at first cycle (P = 0.047). These results shown an increased risk of neutropenia in patient with UGT1A1*28 genotype. The prevalence of UGT1A1*6 in Asian is higher than Caucasian population. The UGT1A1*6 polymorphism is the most common allele that is correlated with reduced SN-38 glucuronidation activity and drug toxicity 26 . Han et al. 27 demonstrated that UGT1A1*6/*6 was significantly associated with higher SN-38 AUC and may increase the risk for toxicities. Onoue et al. 28 performed a prospective study of 135 Japanese cancer patients treated with irinotecan, and found that severe neutropenia was highly correlated with homozygous UGT1A1*6 in a multiple logistic regression analysis. Similarly, UGT1A1*6 was significantly associated with grade 1-4 and severe neutropenia at the first and second cycles in this study. In contrast, there were significant differences between grade 1-4 neutropenia and patients with UGT1A1*28 at the second cycle. However, UGT1A1 genotype was associated with an increased risk of grade 1-4 and severe neutropenia at the first and second cycle. Similar to the study by Yang et al. 29 , UGT1A1*28 and *6 were significantly associated with higher incidence of grade 3-4 neutropenia. www.nature.com/scientificreports/ A meta-analysis by Han et al. 30 found that Asian cancer patients with UGT1A1*28 and *6 are at increased risk of irinotecan-induced neutropenia. Moreover, we also found an association between UGT1A1*28 or/and UGT1A1*6 and ANC nadir. This revealed that patients carrying variant of UGT1A1 genotype had a significantly lower ANC nadir in the first and second cycle. Moriya et al. 31 reported that ANC nadir in patients carrying UGT1A1*6/*28, *6/*6 were significantly lower compared with those with *1/*1. ABCC2 protein is expressed in liver, kidney, and small intestine, and also plays a primary role in biliary excretion of irinotecan and its metabolites 32,33 . Interestingly, our result suggested that ABCC2 c.3972C > T is associated with grade 1-4 neutropenia at the first cycle. Multivariate analysis indicated that ABCC2 c.3972C > T is a risk factor for the occurrence of grade 1-4 neutropenia at the first cycle in patients who receive irinotecanbased chemotherapy. Innocenti et al. 16 reported that ABCC2 c.3972T/T genotype correlates with higher AUC of irinotecan, APC, and SN-38G. This result suggests that ABCC2 c.3972C > T is associated with decreased hepatobiliary excretion of irinotecan and its metabolites.
ABCB1 c.3435C > T is associated with significantly lower AUC SN-38G levels, and homozygous ABCB1 c.3435T/T may be related to higher P-glycoprotein (MDR1) activity 34 . However, ABCB1 c.3435C > T was not associated with irinotecan induced severe neutropenia and diarrhea in Chinese cancer patients who received irinotecan chemotherapy 35 . Cote et al. 36 reported that no statistically significant difference was found in ABCB1 c.3435C > T polymorphism and occurrence of severe hematologic toxicity or severe neutropenia.
The ABCB1 c.1236C > T has been reported to be associated with increased AUC of irinotecan and SN-38 in Caucasian cancer patients 13 , and ABCB1 c.1236T/T had significantly higher plasma irinotecan and SN-38 concentrations than C/C or C/T. However, Han et al. 34 reported that no significant effect of ABCB1 c.1236C > T on irinotecan or its metabolites concentrations. Han et al. 27 reported that no significant association between ABCB1 c.1236C > T and severe neutropenia and diarrhea was observed. In vitro studies have shown that ABCG2, an efflux drug transporter, had a higher affinity with SN-38 and SN-38G 37 , and ABCG2 c.421C > A is related with reduced expression of ABCG2 protein and transporter activity. However, de Jong et al. 38 reported that no significant changes in irinotecan pharmacokinetics relative to the ABCG2 c.421C > A in Caucasian cancer patients. ABCG2 variants had no effect on SN-38 exposure or ANC nadir in 78 irinotecan-treated patients 39 .
Irinotecan is converted by CYP3A4/5 to APC metabolite in the liver, and correlation between these genes and irinotecan induced-toxicity found in this study may be due to low allele frequency of CYP3A4*1B and *18 in sampled population 40 . Similarly, Han et al. 34 did not find any significant association between CYP3A5*3 and toxicity.
The 5-FU-based regimen may cause neutropenia, however, over 80% of 5-FU is metabolized by dihydropyrimidine dehydrogenase (DPD) in the liver 41 . DPYD variants may be related to severe 5-FU-associated toxicities. DPYD*2A and c.1774 C > T variants were not found in this study. Even though, the variant allele of DPYD*5 had a frequency of 0.19, there was no association between DPYD*5 and hematological toxicities.
A retrospective study design and small sample size are limitations of this study. A prospective study involving larger numbers of patients should confirm our study hypothesis. Secondly, rare genetic variants and multiple genes play a role in the irinotecan pathway. Those variants were not considered in our study. Lastly, non-hematologic toxicity (especially severe diarrhea) was not assesses in our study.
In conclusion, combination of UGT1A1*28 and *6 and ABCC2 c.3972C > T genotype are associated with the occurrence of grade 1-4 and severe neutropenia in Thai patients with metastatic colorectal cancer who receive irinotecan-based chemotherapy. Our findings suggest that UGT1A1 genotype and ABCC2 c.3972C > T might be an important predictor for irinotecan induced-toxicity.