Predictive Value of ABCC2 and UGT1A1 Polymorphisms on Irinotecan-Related Toxicities in Patients with Cancer

Abstract

Background:

There is extensive interindividual variability in response and tolerance to anticancer drugs. This heterogeneity provides a major limitation to the “rational” use of cytotoxic drugs, and it becomes a major problem in oncology giving a narrow therapeutic window with a vital risk. Among these anticancer drugs, irinotecan can cause dose-limiting toxicities, commonly diarrhea and neutropenia. Interaction among pathways of activation/inactivation (UGT1A1) and hepatobiliary transport of irinotecan and its metabolites could, in part, explain its interindividual variability. The objective of this study was to perform an exploratory analysis to evaluate the correlation between the genetic polymorphisms of UGT1A1 and ABCC2 with the different toxicities associated with irinotecan treatment.

Materials and Methods:

Seventy-five patients with solid cancers were included, all were administered an irinotecan-based regimen in both Mission Bay Medical Center; and Zuckerberg San Francisco General Hospital from May 2016 to December 2016. The patients' genotyping was performed for both the UGT1A1*28 polymorphism, and the ABCC2 − 1549G>A, and ABCC2 − 1249G>A single nucleotide polymorphism. Comparisons among qualitative data were assessed using the χ²-test, and Fisher's exact test in the case of small group sizes.

Results:

Diarrhea was observed in 40 patients (53.3%), among them only 9 patients had high grades diarrhea (grades III and IV). Grades III/IV of nausea were more frequently associated with the ABCC2-1549 AA genotype (83.3% p = 0.004) in patients with colorectal cancer. In pancreatic cancer, a significant absence of diarrhea grades III-IV was noted in patients with the ABCC2 1249 GG genotype compared to the other ABCC2 1249 genotypes.

Introduction

From a clinical point of view, we find that for the same treatment regimen and in the same clinical situation, some patients respond exceptionally, while others very little or not at all, proving that we are not all the same. Therefore, the human body remains the perfect laboratory, reflecting how differently human cells would, in vivo, react, metabolize, and excrete the drug from the human body. This clarifies how insufficient the premarket testing approaches are in predicting the patient's response and tolerance to candidate therapeutics.

Thus, anticancer drugs make no exception and the response is very variable from one individual to another, both pharmacologically (efficacy sought) and in terms of toxicities (adverse effects). While the variability of this response, which is often difficult to predict, is already an important limitation to the “rational” use of cytotoxic drugs, it becomes a major problem in oncology, giving a narrow therapeutic window with a vital risk. The rationalization of chemotherapy treatments is therefore crucial in this context.

Irinotecan is one of the cytotoxic drugs that have a narrow therapeutic index and could cause side effects, commonly two dose-limiting toxicities, diarrhea and neutropenia (Douillard et al., 2000; Mathijssen et al., 2001; Ratain, 2002), with up to 34% of patients experiencing grade III-IV neutropenia (Fuchs et al., 2003). Therefore, it is more sensitive to metabolic variations, and this motivated the search for genetic biomarkers to safely administer irinotecan and rationalize its use.

Irinotecan displays an extremely complex pharmacological profile, due to the number of enzymes involved in its metabolism and elimination. In humans, irinotecan gets converted to an active form SN-38 by carboxylesterases. SN-38 is 100-1000 times more cytotoxic than irinotecan itself (Mathijssen et al., 2001) by inhibiting the activity of DNA topoisomerase Ι (Topo Ι) (Xu and Villalona-Calero, 2002). Metabolic pathways for irinotecan in humans have been characterized. An irreversible ternary complex of SN-38, DNA topoisomerase Ι enzyme, and the ligated DNA strand is formed, and thus induces a stop of replication and transcription of DNA leading to cell death (Redinbo et al., 1998; Stewart, 1998).

Besides being hydrolyzed, irinotecan is also oxidized to other quantitatively abundant inactive catabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC), the major inactive oxidation product (Mathijssen et al., 2001), and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC), formed by the cytochrome P450 3A4 (CYP3A4) enzyme (Innocenti et al., 2001). These two oxidative metabolites with no known pharmacological activity and being not directly linked to cytotoxic effect are thereby indirectly impacting the availability of irinotecan (CPT-11) and its amount for conversion to SN-38 (Mathijssen et al., 2001). Subsequently, SN-38 undergoes a glucuronidation mediated by uridine diphosphate glucuronosyl transferase (UGT) family members, mainly UGT1A1, the principal pathway of SN-38 detoxification, forming SN-38 glucuronide (Di Paolo et al., 2011).

Compared to the wild-type allele UGT1A1*1, UGT1A1*28 polymorphism, the most common variant allele, has been confirmed to be associated with irinotecan toxicity, specifically neutropenia and diarrhea (Ando et al., 2000; Iyer et al., 2002), due to reduced formation of SN- 38G, and higher or prolonged exposure of SN-38, leading to variability in pharmacokinetics (PKs) of SN-38 and in the toxicity of irinotecan (Innocenti et al., 2004; Toffoli et al., 2006).

Furthermore, the area under curve of SN-38G/SN-38 ratio has been shown to be a clinically influencing factor in CPT-11-induced toxicity (Glimelius et al., 2011; Hirose et al., 2012). This has led to an FDA-mandated label change in August 2005 (Innocenti and Ratain, 2006) and approval of the Invader Molecular Assay for irinotecan dosing, a milestone in the incorporation of pharmacogenomics into routine clinical practice.

Interaction among pathways of activation/inactivation (UGT1A1) and hepatobiliary transport of irinotecan and its metabolites could, in part, explain the interindividual variability aforementioned. Abnormally increased biliary index and SN-38/SN-38glu ratio and high systemic exposure to SN-38 have been demonstrated in patients homozygous for the UGT1A1*28 allele (TA7 allele) with high risk for irinotecan-induced severe diarrhea and neutropenia (Ando et al., 2000; Innocenti et al., 2004), compared to patients harboring at least one wild-type allele (TA6 allele) (Ando et al., 1998; Iyer et al., 2002).

Investigations of adenosine triphosphate (ATP)-binding cassette (ABC) active transporter genes, ABCB1 and ABCC2 in particular (Chu et al., 1998; Chu et al., 1997; Nakatomi et al., 2001), involved in retention and efflux of irinotecan and SN-38 have demonstrated their implication in irinotecan toxicity occurrence, being as essential factors in drug PKs.

Altogether, this supports strongly the benefit of having the genotypes of UGT1A1*28 and active transporter (ABCC2), before dosing as predictive biomarkers of irinotecan induced-toxicity.

In this context, the objective of this study was to perform an exploratory analysis to evaluate the correlation of UGT1A1 and ABCC2 genetic polymorphisms to different toxicities of irinotecan treatment in patients with cancer.

Materials and Methods

Patients

In this study, 75 patients were included, all suffered from a cancer histologically proven and administered an irinotecan-based regimen in both Mission Bay Medical Center, and Zuckerberg San Francisco General Hospital, from May 2016 to December 2016. For inclusion criteria, patients had to be between 18 and 85 years of age, with a performance status ≥2, evaluated as defined by the World Health Organization (WHO). The study was reviewed and approved by the University of California, San Francisco, Institutional Review Board (IRB). Blood samples collected for routine medical purposes were retained for this research study.

The sampling was performed for each patient undergoing irinotecan-based chemotherapy, and blood samples were collected 20 days (500 h) after irinotecan infusion. Approximately 5 mL of blood sample was taken into a heparinized tube. This was scheduled for bioanalytical purpose with liquid chromatography tandem mass spectrometry.

A medical record review was performed on all enrolled subjects. The IRB determined that this protocol represented low risk; thus, patients were not consented.

Patient genotyping: genotyping of *UGT1A1**28 polymorphism, ABCC2 − 1549G>A and ABCC2 − 1249G>A single nucleotide polymorphism (SNPs)

Genomic DNA was isolated from 200 μL of whole blood, which had been stored at −80°C until analysis, using QIAamp^® DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions. The DNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). TA repeats in the UGT1A1 promoter, and variations in ABCC2 at positions 1549G>A and 1249G>A SNPs were genotyped using real-time polymerase chain reaction (PCR) and BHQ Plus probes (Biosearch Technologies, Novato, CA). The PCR reactions were performed using the Rotor-Gene Q real-time PCR system (Qiagen, Inc., Valencia, CA). UGT1A1 sequences were designed by Ehmer et al. (2008). ABCC2 primer and probe sequences were designed using Real Time Design software (Biosearch Technologies) and are shown in Supplementary Table S1.

The PCR mixture was (15 μL): 7.5 μL of Master mix, 0.75 μL of probe/primer mix (100 ng/mL), 2 μL of DNA template, and 4.75 μL of double distilled water. UGT1A1 amplification procedure was as follows: an initial polymerase activation at 95°C for 20 s (TaqMan GTxpress Master Mix), followed by 50 cycles of denaturation at 95°C for 5 s, annealing (55°C for 15 s), and extension (64°C for 30 s). UGT1A1 genotypes were assigned as *1/*1, *1/*28, *28/*28, where *1 represents the reference allele containing six TA repeats (UGT1A1*1), whereas *28 represents the variant allele containing seven TA repeats (UGT1A1*28), respectively. For quality control purposes, wild-type, heterozygous, and homozygous variant sequenced samples were included in each run. The results were analyzed using Rotor Gene 2.1 software.

For ABCC2 1249 and 1549, amplification procedure was as follows: an initial polymerase activation at 95°C for 10 min (TaqMan Universal Master Mix), followed by 40 cycles of denaturation at 95°C for 15 s, annealing (60°C for 1 min), and finally extension at 60°C for 1 min. ABCC2 haplotypes were assigned as ABCC2*2 when −1549 G and 1249G and as ABCC2*3 when −1549 G and 1249A, and as “other” for the other remaining haplotypes.

Statistical analysis

Statistical analysis was assessed by SPSS (Statistical Package for the Social Sciences) 22.0 software (IBM Corporation, Armonk, NY). Descriptive variables were expressed as mean ± standard deviation or medians (interquartile range). Comparison between qualitative data was assessed by the Chi-square test, and Fisher's exact test in case of low group sizes. The p-value was considered significant when p < 0.05.

Results

Clinical characteristics

Patients' clinical characteristics are shown in Supplementary Table S2. This study enrolled 33 females and 42 males and the mean age was 53.6 ± 12.7 years. Colorectal cancer was the primary tumor localization followed by pancreatic cancer in 54.7% and 32% of patients, respectively. Of the study population, 56% were Caucasian, 22.7% were Asian, and only 5.3% were African American. More than half of patients (60%) were nonsmokers, while 33.3% were former smokers. Nearly three quarters of our patients (74.7%) did not have alcohol history. There were 48 patients (68.8%) who had metastatic disease at the time of diagnosis and often metastases were detected in only 1 site (60.4%). KRAS and BRAF status were screened in few patients. Thirteen out of thirty-five patients had mutant KRAS and only 2/17 patients had mutant BRAF.

Irinotecan therapy and toxicities

Among all the patients, 92% had palliative treatment, and the most used chemotherapy treatment was irinotecan combined to other molecules (96%). FOLFIRI (irinotecan, leucovorin, and 5-fluorouracil) regimen was administered to 27 patients (36%) and FOLFIRINOX (addition of oxaliplatin) to 24 patients (32%). The different toxicities attributable to irinotecan in patients, namely neutropenia, diarrhea, nausea, and vomiting, were recorded in this study. There were 59 patients (78.7%) who had developed at least 1 toxicity and nearly half of these patients (49.3%) had experienced more than one type of toxicity during irinotecan therapy (Table 1).

Table 1.

Chemotherapy Regimen and Toxicities

Characteristics	No. of patients (n = 75), %
Chemotherapy regimens
Monotherapy (Irinotecan)	3 (4.0)
Combination	72
FOLFIRI	27 (36.0)
FOLFIRINOX	24 (32.0)
FOFIRI+ monoclonal antibody	10 (13.3)
Others	11 (14.7)
Toxicity (treatment outcomes)
Overall toxicity
Yes	59 (78.7)
No	16 (21.3)
One type toxicity	22 (29.3)
1+ Toxicity	37 (49.3)
Grade of toxicities
G III-IV	17 (22.7)
Early	23 (30.7)
Types of toxicity/grade
Diarrhea G I-II	31 (41.3)
Diarrhea G III-IV	9 (12.0)
Nausea G I-II	37 (49.3)
Nausea G III-IV	12 (16.0)
Vomiting G I-II	10 (13.3)
Vomiting G III-IV	5 (6.70)
Neutropenia G I-II	3 (4.00)
Neutropenia G III-IV	4 (5.30)
Dose reduction/delay/discontinuation/withdrawal (due to toxicity)
Irinotecan dose reduction	11 (14.7)
Premature discontinuation (ending treatment)	03 (4.00)
Delay in administration (interruption treatment)	09 (12.0)
Withdrawal treatment (irinotecan withdrawn from regimen)	02 (2.70)

Diarrhea was observed in 40 patients (53.3%); among them, only 9 patients had high-grade diarrhea (grades III and IV). The incidence of nausea, vomiting, and neutropenia was 65.3% (n = 49), 20% (n = 15), and 9.3% (n = 7), respectively. Due to severe toxicities, 11 patients had to have dose reduction of irinotecan, while 3 patients ended treatment prematurely.

Frequency of UGT1A1 genotypes and ABCC2 variant

Genotyping results are listed in Table 2. All SNP frequencies are in Hardy-Weinberg equilibrium. The number of TA repeats in the TATA-box of the UGT1A1 gene promoter region was successfully determined in 75 patients. Three groups of the UGT1A1 genotype were constructed: TA6/6 (wild type), TA6/7 (heterozygous mutant), and TA7/7 (homozygous mutant). The frequency of the UGT1A1*28 allele was found to be 28%. Two variants in the ABCC2 gene were studied in this population. The allele frequencies of 1549G>A and 1249G>A were 0.44 and 0.37, respectively. Frequencies of haplotypes were 0.487 and 0.067 for ABCC2*2 and ABCC2*3, respectively. More details are shown in Table 2.

Table 2.

Distribution of UGT1A1^*28 Genotype and ABCC2 Investigated Polymorphisms and Haplotypes

Genotype	No. of patients (n = 75), %
UGT1A1
6/6	38 (50.7)
6/7	32 (42.7)
7/7	5 (6.7)
ABCC2
−1549
G/G	29 (38.7)
G/A	25 (33.3)
A/A	21 (28.0)
1249
G/G	58 (77.3)
G/A	15 (20.0)
A/A	2 (2.7)
ABCC2 haplotype
^2/^2	23 (30.7)
^*2/Other	27 (36.0)
^3/^3	2 (2.7)
^*3/Other	6 (8.0)
Other	17 (22.7)

Toxicity-genotype relationship

There was a significant difference on vomiting grades I/II and grades III/IV in patient groups with different ABCC2-1549 genotypes with p-values of 0.028 and 0.045, respectively (Table 4). For the ABCC2-1249 genotypes, no significant difference was found with any of the different types of toxicities (Tables 3 and 4). Besides, ABCC2 haplotypes showed significant difference of nausea grades III/IV occurrence (Table 4) across different groups (p = 0.009).

Table 3.

Relationship Between UGT1A1 and ABCC2 Gene Polymorphism and Toxicity Occurrence

Genotyping	Toxicity occurrence		p
Genotyping	Yes, n (%)	No, n (%)	p
UGT1A1
WT (TA6/6)	29 (49.2)	9 (56.3)
Heterozygous (TA6/7)	27 (45.8)	5 (31.3)	0.321
Mutant (TA7/7)	3 (5.1)	2 (12.5)
ABCC2- 1549
WT (G/G)	21 (35.6)	8 (50.0)
Heterozygous (G/A)	21 (35.6)	4 (25.0)	0.565
Mutant (A/A)	17 (28.8)	4 (25.0)
ABCC2 1249
WT (G/G)	44 (74.6)	14 (87.5)
Heterozygous (G/A)	13 (22.2)	2 (12.5)	0.690
Mutant (A/A)	2 (3.4)	0 (0.0)
ABCC2 haplotype
^2/^2	16 (27.1)	7 (56.3)
^*2/Other	21 (35.6)	6 (37.5)	0.617
^3/^3	2 (3.4)	0 (0.0)
^*3/Other	6 (10.2)	0 (0.0)
Other	13 (28.0)	3 (18.8)

WT, wild type.

Table 4.

Relationship Between UGT1A1 ^*28 Genotypes and ABCC2 Genotype for Investigated Polymorphisms and Toxicities Related to Irinotecan (CPT-11)

Genotype	Neutropenia, n (%)		Diarrhea, n (%)		Nausea, n (%)		Vomiting, n (%)
Genotype	G I-II	G III-IV	G I-II	G III-IV	G I-II	G III-IV	G I-II	G III-IV
UGT1A1
6/6	1 (33.3)	3 (75.0)	15 (48.4)	4 (44.4)	22 (59.5)	3 (25.0)	5 (50.0)	2 (40.0)
6/7	2 (66.7)	1 (25.0)	15 (48.4)	5 (55.6)	13 (35.1)	8 (66.7)	5 (50.0)	3 (60.0)
7/7	0 (0.0)	0 (0.0)	1 (3.2)	0 (0.0)	2 (5.4)	1 (8.3)	0 (0.0)	0 (0.0)
p	0.667	0.713	0.540	0.856	0.298	0.114	1.000	0.758
ABCC2
−1549
G/G	2 (66.7)	2 (50.0)	14 (45.2)	4 (44.4)	14 (37.8)	2 (16.7)	6 (60.0)	0 (0.0)
G/A	1 (33.3)	2 (50.0)	10 (32.3)	2 (22.3)	15 (40.5)	3 (25.0)	0 (0.0)	4 (80.0)
A/A	0 (0.0)	0 (0.0)	7 (22.6)	3 (33.3)	8 (21.6)	7 (58.3)	4 (40.0)	1 (20.0)
p	0.775	0.549	0.557	0.742	0.334	0.055	0.028	0.045
1249
G/G	1 (33.3)	4 (1.00)	22 (71.0)	5 (55.6)	29 (78.4)	9 (75.0)	9 (90.0)	4 (80.0)
G/A	2 (66.7)	0 (0.0)	8 (25.8)	3 (33.3)	6 (16.2)	3 (25.0)	1 (10.09)	1 (20.0)
A/A	0 (0.0)	0 (0.0)	1 (3.2)	1 (11.1)	2 (5.4)	0 (0.0)	0 (0.0)	0 (0.0)
p	0.543	0.619	0.545	0.99	0.359	0.790	0.757	1.000
ABCC2 haplotype
^2/^2	1 (33.3)	2 (50.0)	10 (32.3)	0 (0.0)	11 (29.7)	1 (8.3)	5 (50.0)	0 (0.0)
^*2/Other	2 (66.7)	2 (50.0)	11 (35.5)	5 (55.6)	17 (45.9)	2 (16.7)	1 (10.0)	3 (60.0)
^3/^3	0 (0.0)	0 (0.0)	1 (3.2)	1 (11.1)	2 (5.4)	0 (0.0)	0 (0.0)	0 (0.0)
^*3/Other	0 (0.0)	0 (0.0)	3 (9.7)	0 (0.0)	2 (5.4)	2 (16.7)	0 (0.0)	1 (20.0)
Other	0 (0.0)	0 (0.0)	6 (19.7)	3 (33.3)	5 (13.5)	7 (58.3)	4 (40.0)	1 (20.0)
p	0.844	0.792	0.964	0.057	0.116	0.009	0.170	0.360

Bold values are significant p-values.

Race was also investigated and is summarized in Table 5. We found that ABCC2-1549 shows significant correlation with race. Caucasian patients had more A/A genotype (40.5%), African American had more G/A genotype (60%), and Asian were more G/G genotype (64.7%); p-value of 0.011. As for UGT1A1 (TA)n/n variants, no statistically significant difference was noticed between different races of our study population. In Supplementary Table S4, we attempted to compare, into each UGT1A1 genotype group (6/6; 6/7; 7/7), the occurrence of toxicity (types/grades) between different ABCC2 haplotype subgroups. No significant difference was found.

Table 5.

Distribution of UGT1A1^*28 and ABCC2 Genotypes in Patients with Different Races

Genotyping	Race				p
Genotyping	Caucasian	African American	Asian	Other	p
UGT1A1
6/6	18 (42.9)	2 (50.0)	12 (70.6)	6 (50.0)
6/7	20 (47.6)	2 (50.0)	5 (29.4)	5 (41.7)	0.583
7/7	4 (9.5)	0 (0.0)	0 (0.0)	1 (8.3)
ABCC2 − 1549
G/G	13 (31.0)	0 (0.0)	11 (64.7)	5 (41.7)
G/A	12 (28.6)	4 (60.0)	5 (29.4)	4 (33.3)	0.011
A/A	17 (40.5)	0 (0.0)	1 (5.9)	3 (25.0)
ABCC2 1249
G/G	32 (76.2)	3 (75.0)	14 (82.4)	9 (75.0)	0.851
G/A	9 (21.4)	1 (25.0)	3 (17.6)	2 (16.7)
A/A	1 (2.4)	0 (0.0)	0 (0.0)	1 (8.3)
ABCC2 haplotype
^2/^2	10 (23.8)	0 (0.0)	9 (52.9)	4 (33.3)
^*2/Other	13 (31.0)	3 (75.0)	7 (41.2)	4 (33.3)
^3/^3	14 (33.3)	0 (0.0)	2 (16.7)	2 (16.7)	0.095
^*3/Other	1 (2.4)	0 (0.0)	0 (0.0)	1 (8.3)
Other	4 (9.5)	1 (25.0)	0 (0.0)	1 (8.3)

Bold value is significant p-value.

We studied the differences between sex (male and female) and different genotypes (polymorphisms distribution [UGT1A1 and ABCC2]), and between sex (male and female) in the occurrence of grades of nausea and vomiting. The statistic test (Chi-square) did not reveal a significant difference, and the comparison parameters showed a p > 0.05. Results are shown in Supplementary Tables S5 and S6, respectively.

We also studied the differences in sex (male and female) for the occurrence of toxic events (grades of neutropenia, diarrhea, nausea, and vomiting), correlated to polymorphisms. The Chi-square statistic test showed significant differences on this behalf.

According to Table 6, in males, grades III/IV of vomiting are more frequent in males when ABCC2-1549 genotype is G/A (n = 4; 100%) with significant p-value (p = 0.007), and grades III-IV of diarrhea are more frequent when ABCC2 1249 genotype is G/A (n = 2; 50% p = 0.032). Males with UG1A1 6/7 are more exposed to grades III/IV of nausea (n = 6; 85.7%), p = 0.006.

Table 6.

Relationship Between UGT1A1^*28 and ABCC2 Genotypes, and the Incidence of Toxicities (Types and Grades) in Patients According to Sex

Sex	Specific genotype	Neutropenia, n (%)		Diarrhea, n (%)		Nausea, n (%)		Vomiting, n (%)
Sex	Specific genotype	G I-II	G III-IV	G I-II	G III-IV	G I-II	G III-IV	G I-II	G III-IV
M (n)	ABCC2
	−1549
	G/G	1	1	9	3	9	1	2	0
	G/A	1	0	3	0	5	2	0	4
	A/A	0	0	6	1	7	4	2	0
p		0.58	0.39	0.14	0.18	0.52	0.28	0.37	0.007
F (n)	−1549
	G/G	1	1	5	1	5	1	4	0
	G/A	0	2	7	2	10	1	0	0
	A/A	0	0	1	2	1	3	2	1
p		0.49	0.45	0.15	0.42	0.007	0.07	0.12	0.14
M (n)	1249
	G/G	1	1	12	1	15	6	4	3
	G/A	1	0	5	2	4	1	0	1
	A/A	0	0	1	1	2	0	0	0
p		0.58	0.83	0.65	0.032	0.34	0.68	0.45	0.88
F (n)	1249
	G/G	1	3	10	4	14	3	5	1
	G/A	0	0	3	1	2	2	1	0
	A/A	0	0	0	0	0	0	0	0
p		0.63	0.39	0.55	0.9	0.41	0.17	0.91	0.63
	UGT1A1
M (n)	6/6	1	1	8	2	11	0	1	1
	6/7	1	0	9	2	8	6	3	3
	7/7	0	0	1	0	2	1	0	0
p		0.83	0.62	0.19	0.67	0.87	0.006	0.21	0.21
F (n)	6/6	0	2	7	2	11	3	4	1
	6/7	1	1	6	3	5	2	2	0
	7/7	0	0	0	0	0	0	0	0

In females, grades I-II of nausea are more frequent when ABCC2-1549 genotype is G/A (62.5%) with significant p-value (p = 0.007) or when UGT1A1 genotype is 6/6 (n = 11; 68.6%) (p = 0.024). Grades III-IV of nausea are more frequent when ABCC2-1549 genotype is A/A (n = 3; 60.0%; p = 0.07).

The statistic test revealed no significant difference for the other toxicities and genotypes in males and females, and the comparison parameters showed a p > 0.05.

Discussion

This is a pharmacogenetic investigation that confirms the importance of prior knowledge of the genotype of UGT1A1*28 and ABCC2, as predictive biomarkers of irinotecan-induced toxicity.

In our cohort, we studied the relationship between UGT1A1 TA repeats polymorphism and ABCC2 variants/haplotypes and different CPT-11-related adverse reactions (ADR), including neutropenia, diarrhea, nausea, and vomiting.

In the coming paragraphs, we are discussing the interindividual predisposition of patients to irinotecan ADR above, due to genetic polymorphism predisposition.

Frequency of UGT1A1 genotypes and ABCC2 variants

From the 75 patients in whom UGT1A1 genotypes were determined, three groups of UGT1A1 genotype were identified: TA6/6 (wild type), TA6/7 (heterozygous mutant), and TA7/7 (homozygous mutant). The allele frequency of the UGT1A1*28 gene was 28%. The genotype distribution among 75 patients enrolled in this study was as follows: 38 patients (50.7%) with the TA6/6 genotype (*1/*1) and 32 patients (42.7%) with the TA6/7 genotype (*1/*28), while TA7/7 genotype (*28/*28) was found in only 5 patients (6.7%), which is very similar to the frequencies found by de Jong et al. (2007) (p = 1). However, there was a significant difference with the results reported by Bai et al. (2017) (p = 0.001).

Two variants in the ABCC2 gene were studied in this population (Table 2). The allele frequencies of the −1549G>A and 1249G>A were 0.44 and 0.37, respectively (Table 2). Different frequencies were reported by other population studies. Fujita et al. (2008) reported 0.157 and 0.134 as −1549G>A and 1249G>A respective allele frequencies, while de Jong et al. (2007) reported relative frequencies of 0.4 and 0.022. According to this, we can say that we found the allele frequencies fit between values of allele frequencies found by the studies of Fujita et al. (2008) and de Jong et al. (2007).

For the G-1549A, 29 patients had the G/G (38.7%), 25 had the G/A (33.3%), and 21 patients with the A/A genotypes (28%). A total of 58 patients had the G/G genotype for the ABCC2 1249 G > A (77.3%), 15 with the G/A (20%), and only 2 with the A/A genotypes (2.7%). Compared to a previous study (Fujita et al., 2008), these frequencies are roughly comparable in case of ABCC2 1249 genotypes (p > 0.05). While genotype frequencies of ABCC2 − 1549 seem to be significantly different from ours, notably in case of G/G and A/A genotypes, p-value were respectively 3.77 10⁻⁵ and 0.0001. Two haplotypes were constructed in the 75 patients who had been successfully genotyped for the 2 variants (Table 2). Frequencies of haplotypes were 0.487 and 0.067 for ABCC2*2 and ABCC2*3, respectively. de Jong et al. (2007) and Fujita et al. (2008) reported similar haplotype frequencies.

Toxicity-genotype relationship

To evaluate the phenotype-genotype relationship in our cohort, Table 4 shows a significant difference on vomiting grades I/II and grades III/IV in patient groups with different ABCC2-1549 genotypes. The pairwise comparisons between these groups show that patients with G/A ABCC2-1549 genotype are less likely to develop grade I-II vomiting. Seemingly, patients with G/G ABCC2-1549 genotype are less likely to develop grade III-IV vomiting. These findings may reflect a protective effect of these two genotypes, G/A and G/G, against vomiting toxicity. For the ABCC2 1249 genotypes, no significant difference was found with any of the different types of toxicities (Tables 3 and 4). Contrariwise, in a study by Fujita et al. (2008), 17% (1/6) of patients harboring at least one allele of 1249G>A and 40% (10/25) of those with wild-type genotype had grade 3 or higher neutropenia (p > 0.05).

On the other hand, there was no significant difference on toxicity occurrence (all types and grades) in patients with different UGT1A1*28 and ABCC2 genotypes/haplotypes. A previous study has revealed that patients with at least one UGT1A1*28 allele was 1.3-fold in high risk of severe diarrhea occurrence (95% confidence interval, 0.53-3.13), but this was not statistically significant (p = 0.587). Likewise, UGT1A1*28 genotype was not related to occurrence of neutropenia (p = 0.411) (de Jong et al., 2007). Toffoli et al. (2006) concluded that the relevance to irinotecan toxicity of UGT1A1*28 is limited.

Besides, ABCC2 haplotypes showed significant difference of nausea grades III/IV (p = 0.009) occurrence (Table 4) across different groups (p = 0.009). When comparing these groups by pairs, we found significant difference between the genotype so-called “other/other” (identified in Materials and Methods section) and all the remaining haplotypes (*2/*2; *2/other; *3/*3; *3/other), given that all the patients with “other/other” haplotype have grade III-IV nausea. This means that this particular haplotype is indeed strongly related to high risk of developing nausea during irinotecan exposure, that is, we showed that the remaining haplotypes provide a protective effect against nausea occurrence for the carriers.

Supplementary Table S3 summarizes relationship between UGT1A1*28 and ABCC2 genotypes/haplotypes and toxicities in patients with different tumor locations. It shows that in patients with colorectal cancer, grades III/IV of nausea are more frequent when ABCC2-1549 genotype is A/A (83.3%) with significant p-value (p = 0.004). ABCC2 haplotypes are more likely to be correlated with nausea and vomiting. In fact, grade I/II nausea is more frequent in “2*/other” haplotype (47.8%), while grade III/IV nausea is more frequent in “3*/other” haplotype (83.3%) with significant p-values (Table 4). de Jong et al. (2007) found that ABCC2*2 allele was not significantly related with neutropenia (p = 0.96), or less severe diarrhea (odds ratio, 0.55; 95% confidence interval, 0.22-1.34; p = 0.185).

Comparing colorectal cancer and lung cancer patients, Yui et al. (Ando et al. 2000) found in lung cancer group, a high risk of grade I/II neutropenia among patients with UGT1A1*28 mutant genotypes (18.18%; 2/11) compared to the wild-type genotype patients (5.88; 2/34), but this difference was not statistically significant (p = 0.247). Thus, no significant association of both genotypes (UGT1A1*28 mutant or wild type) with severe toxicities was found. However, in the colorectal cancer group patients with mutant UGT1A1*28 genotypes, 28.57% (2/7) were in high risk of severe delayed diarrhea compared to patients with wild-type genotype (10.00%; 2/20), even if this difference was not statistically significant (p = 0.269) (Ando et al. 2000).

Race was also investigated and results are summarized in Table 5. We found that ABCC2-1549 shows significant correlation with race. Caucasian patients had more A/A (40.5%), African American had more G/A (60%), and Asian were more G/G genotypes (64.7%), with a p-value of 0.011. As for UGT1A1 (TA)n/n variants, no statically significant difference was noticed between different races of our study population. In a series of U.S. colorectal cancer patients, the variant frequencies were 43% for the (TA)6/6 genotype, 48% for the (TA)6/7 genotype, and 9% for the (TA)7/7 genotype (Iyer et al. 2002).

Among race groups, the (TA)7/7 genotype is commonly present in African descent and rare in Asian descent, as the high frequency is among African Americans (0.38-0.45), followed by Caucasians (0.29-0.39), and much lower in Asians (0.02-0.14) (Beutler et al., 1998; Kaniwa et al., 2005). However, no significant difference was noticed for UGT1A1 TA repeat genotypes regarding race, reflecting a homogenous distribution of all genotypes across the studied population (p > 0.05).

In Supplementary Table S4, we correlated, into each UGT1A1 genotype group (6/6; 6/7; 7/7), the occurrence of toxicity (types/grades) to different ABCC2 haplotype subgroups. No significant difference was noticed. However, in a previous study (de Jong et al., 2007), when both UGT1A1*28 genotype and ABCC2*2 haplotype were considered together, patients harboring UGT1A1*1/*1 (TA6/6) genotype and ABCC2*2 haplotype were protected against severe diarrhea (10 vs. 44%; odds ratio, 0.15; 95% confidence interval, 0.04-0.61; P1/40.005). This effect is not observed in patients heterozygous or homozygous for UGT1A1*28 variant (32% vs. 20%; odds ratio, 1.87; 95% confidence interval, 0.49-7.05; P1/40.354).

In our series, no significant difference was noted in polymorphism distribution of neither UGT1A1 nor ABCC2 genotypes according to sex. A recent study (Bandyopadhyay et al., 2021) has noted same results reporting no significant difference in the distribution of UGT1A1*28 genotypes between males and females with a p-value = 0.99.

We studied the differences between sex in the occurrence of grades of nausea and vomiting, but no significant difference was found, whereas Liu et al. (2017) has studied neutropenia and diarrhea with sex and found that the severe neutropenia incidence was 24.7% in females, and 18.0% in males, with p-value of 0.056, but no difference was found in diarrhea between sex.

Certainly, in the context of pilot experiments such as this study, these observations hold their importance. Besides, it should be noted that more confirmatory investigations of these observations, with a quiet larger population size, are warranted and become necessary.

Being a gene encoding for an important enzyme involved in phase II conjugation pathway, UGT1A1*28 polymorphism affects CPT-11 metabolism and detoxification of its active metabolite, SN-38, leading to toxicity, in particular, neutropenia. However, as a practical matter for oncologists and clinicians, guidelines on dose adjustment for patients carrying this polymorphism are unclear. Pre-therapeutic UGT1A1*28 screening does not identify all individuals at high risk of irinotecan-related diarrhea.

Previous studies (de Jong et al., 2007; Toffoli et al., 2006) concluded that UGT1A1*28 is of limited relevance as a predictor biomarker of toxicity following irinotecan treatment. This may be explained by the need of given other genes, such as ABCC2, in hepatobiliary secretion of CPT-11 and SN-38, as well as SN-38G (Sugiyama et al., 1998). Atasilp et al. (2020) has recently reported that UGT1A1*28, UGT1A1*6 (c.211G>A), or ABCC2 c.3972C>T variant might be an important predictor for irinotecan-induced severe neutropenia. ABCC2 gene encodes a membrane protein endogenously expressed at highest levels in the hepatocyte, in addition to kidneys, and the intestines (Ito et al., 2005), enabling transport of various substrates across cell membranes.

Numerous ABCC2 polymorphic variants have been described (Ito et al., 2005; Ito et al., 2001), particularly ABCC2 G-1549A and ABCC2 G1249A (Kroetz et al., 2006). Altered functionality caused by inherited variability of ABCC2 leads to decreased transport of irinotecan into the bile. This is frequently compensated by other transporters, ABCB1 or ABCG2, which compensate for the transport of irinotecan, SN-38, and SN-38G into the bile, or by ABCC1, which transports the irinotecan back into the circulation.

It is worth noting the impact of relative affinities of transporters to drive SN-38, SN-38G, or irinotecan (de Jong et al., 2007). Seemingly, ABCC2 fluxes preferentially the non-glucuronide form of SN-38. Given that diarrhea is caused by increased hepatobiliary secretion of SN-38 into the gut through the bile, impaired ABCC2 transporter could prevent this complication from occurring. ABCC2*2 haplotype, one of the haplotypes with wild G allele (ABCC2 1249G), had significantly decreased messenger RNA (mRNA) levels (Kroetz et al., 2006), while the significantly higher mRNA level was found in patients with the variant 1249G>A.

According to de Jong et al. (2007), some patients are protected against irinotecan-related severe diarrhea only in a particular condition, which is the simultaneous harbor of homozygous UG1A1*1 genotype and ABCC2*2 haplotype. This protective effect is explained by the fact that in the presence of UGT1A1*/*1 and ABCC2*2 haplotype, with the assumption that ABCC2*2 haplotype represents impaired transport function, the SN-38G could not be excreted to intestine neither by altered ABCC2 nor by the compensatory activity of the other transporters (ABCB1 and ABCG2), because of their low affinity to the glucuronide form. However, with UGT1A1*28 allele, the toxic SN-38 will be alternatively excreted by the latter transporters above.

In other words, high formation of SN-38G (UGT1A1*1) and low activity to transport the SN-38G formed to the gut might protect one from severe diarrhea, knowing that the latter is directly related to the transport of SN-38G to the gut, where it is hydrolyzed by microbial ß-glucuronidases to SN-38 and becomes locally toxic, leading to diarrhea.

Conclusion

Research in pharmacogenetics of many drugs, especially antineoplastic agents, has explained their PK variability among patients, highlighting the role of genetic disparities and polymorphisms. Irinotecan, as an anticancer drug, causes toxicities, notably neutropenia and diarrhea, outweighing the cytotoxic effect desired and limiting its clinical application. UGT1A1 and ABCC2 are two genes encoding for two key proteins that play an important role in SN-38 disposition, the active metabolite of UGT1A1. The different UGT1A1*28 and ABCC2 genotypes result in variation of metabolic rates, and consequently elevated SN-38 blood concentrations, inducing ADR. Thus, using UGT1A1*28 and ABCC2 genotyping may predict the likelihood of SN-38-induced toxicities. Currently, there is no uniform conclusion regarding the correlation between both UGT1A1*28 polymorphism and ABCC2*2 haplotype, and the protective effect against severe diarrhea. Through our study, this relationship could not be verified, confirming further the need for additional rigorous investigations.

Irinotecan-related toxicities occur rather frequently. Thus, to define individually predictive measures of their occurrence, additional researches are warranted. The limitation of this study is the small sample size of the population. Owing to the low UGT1A1*28 polymorphism and ABCC2 variant rates, a larger sample size is needed to verify the finding.

Ethical Statement

This study was approved, on April 2016, by the University of California, San Francisco IRB. The UCSF IRB determined that this protocol represented low risk; thus, patients were not consented.

Data Accessibility

The datasets used and analyzed during this study are available from the corresponding author on reasonable request.

Footnotes

Authors' Contributions

Z.A.: conceptualization, methodology, software, data curation, investigation, writing—original draft, writing-review and editing, and project administration. A.S.: methodology and visualization. M.S.: software, formal analysis, and review and editing. I.E.B.: formal analysis and review and editing. H.G.: writing—review and editing. N.A.I.: writing—review and editing. B.M.: supervision. K.L.L.: supervision. Y.C.: supervision and validation. A.H.B.W.: conceptualization, methodology, resources, supervision, writing—review and editing, and project administration.

Author Disclosure Statement

We wish to confirm that there are no potential conflicts of interest associated with this publication.

Funding Information

Z.A. is a Fulbright Scholar. This research did not receive any other specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Supplementary Material

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Supplementary Material

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Predictive Value of ABCC2 and UGT1A1 Polymorphisms on Irinotecan-Related Toxicities in Patients with Cancer

Abstract

Background:

Materials and Methods:

Results:

Introduction

Materials and Methods

Patients

Patient genotyping: genotyping of UGT1A1*28 polymorphism, ABCC2 − 1549G>A and ABCC2 − 1249G>A single nucleotide polymorphism (SNPs)

Statistical analysis

Results

Clinical characteristics

Irinotecan therapy and toxicities

Frequency of UGT1A1 genotypes and ABCC2 variant

Toxicity-genotype relationship

Discussion

Frequency of UGT1A1 genotypes and ABCC2 variants

Toxicity-genotype relationship

Conclusion

Ethical Statement

Data Accessibility

Footnotes

Authors' Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Patient genotyping: genotyping of *UGT1A1**28 polymorphism, ABCC2 − 1549G>A and ABCC2 − 1249G>A single nucleotide polymorphism (SNPs)