Predictive Value of UGT1A1 Polymorphisms in Irinotecan-Induced Toxicity and Therapeutic Efficacy in Colorectal Cancer Patients

Page 39 of 46 Predictive Value of UGT1A1 Polymorphisms in Irinotecan-Induced Toxicity and Therapeutic Efficacy in Colorectal Cancer Patients Qianqian Yu1, Zhihuan Li2, Xiaoqi Nie1, Lu Wang1, Chen Gong1, Bo Liu1, Xin Liao3, Ben Zhao1, Qianxia Li1, Mingsheng Zhang1, Hong Qiu1, Xianglin Yuan1* 1Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China. 2Dongguan Enlife Stem Cell Biotechnology Institute, Dongguan 523000, Guangdong, China 3Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China


Introduction
Colorectal cancer (CRC) is the third most frequent neoplasm and the second leading cause of cancer-related death world-wide 1 . Around 25% of patients present with metastatic or inoperable disease at initial diagnosis, and more than 50% of patients will receive therapeutic regimens involving cytotoxic agents during the course of their illness 2,3 . Chemotherapy consisting of 5-fluorouracil (5-FU) in combination with either oxaliplatin or irinotecan remains the fundamental treatment for metastatic or recurrent CRC. In terms of irinotecan-based schedules, 45-65% of patients failed to respond to treatment and the use of irinotecan is accompanied by a comparably high incidence of severe adverse events [4][5][6][7] . As individualized therapy guided by genotyping has been prevalent, efforts are needed to discover confirmative molecular biomarkers for optimizing and personalizing treatment procedures.
Irinotecan exerts its anti-proliferative cytotoxic effects by inhibiting topoisomerase I required for DNA replication and transcription through active metabolite 7-ethyl-10hydroxycamptothecin . Clinical pharmacological evidence demonstrated that irinotecan-related toxicity and efficacy is associated with objects' exposure to SN38 8 . SN-38 is finally metabolized to an inactive form SN-38 glucuronide (SN38G) by UDPglucuronosyltransferase (UGT)1A enzymes encoded by the UGT1A gene family 9 . The predominant enzyme is UGT1A1, which is also involved in the metabolism of bilirubin 10 . Polymorphisms in UGT1A1 genes may contribute to changes in UGT1A1 enzyme activity, resulting in variability of irinotecan pharmacokinetics 11 . Several UGT1A1 polymorphisms are reported to be associated with irinotecan-related toxicity and efficacy [12][13][14][15][16] . Despite the conflicting results derived from different studies, potential predictive effects of these loci are still promising.

UGT1A1 Polymorphisms Relationship to Toxicity
The risk of irinotecan-induced toxicity, predominantly neutropenia and diarrhea, increases with the polymorphism of genes involved in irinotecan metabolic pathway 17 . Genetic polymorphisms in the UGT1A1 gene, such as UGT1A1*28 and UGT1A1*6, were reported to be associated with decreased UGT1A1 expression or reduced enzymatic activity 18,19 and were usually suggested to be risk factors for severe diarrhea and neutropenia 20 . In the United States, Japan, and some other countries, the recommendation was included in irinotecan product label that a reduction in the starting dose of irinotecan be considered for patients harboring homozygous UGT1A1 *28 (*28/*28 genotype) or *6 (*6/*6 genotype) allele, or heterozygous for both UGT1A1 *28 and *6 alleles (*28/*6 genotype) 21,22 .
Subgroup analysis of the meta-analysis conducted by Zhang et al. 32 31 showed that subjects with UGT1A1*28/*28 genotype had more than fourfold (OR, 4.79; 95% CI, 3.28-7.01) and UGT1A1*1/*28 genotype had approximately twofold (OR, 1.90; 95% CI, 1.44-2.51) increases in the risk of severe neutropenia respectively compared to wild-type genotype; and UGT1A1*28/*28 genotype had an OR of 1.84 (95% CI, 1.24-2.72) for an increased risk of severe diarrhea, while UGT1A1*1/*28 genotype showed no significant correlation with diarrhea. Similar significance persisted in subgroups (high/medium dose or low dose, cutoff value = 150 mg/m 2 ; with 5-FU or without 5-FU) of the analysis between genotypes and neutropenia. The higher incidence of diarrhea in homozygous UGT1A1*28 patients was limited to groups in which irinotecan was given at higher doses or combined with 5-FU. In line with the result of Liu et al., UGT1A1*28 polymorphism was found to be an indicator for neutropenia and diarrhea susceptibility in the meta-analysis by Yang et al. 34 which compromising both Caucasian and Asian trials. The relationship between UGT1A1*28 genotypes and irinotecan-induced toxicity is shown in Table 1.
Our previous study 36 , based on a prospective multicenter longitudinal trial of metastatic CRC patients treated with irinotecan-based therapy, showed that patients carrying UGT1A1*28 allele had more than two-fold higher risk of severe diarrhea compared with UGT1A1*1/*1 patients (OR, 2.673; 95% CI, 1.039-6.876), with an incidence of 11.3% in UGT1A1*1/*1 genotype and 26.2% in patients carrying UGT1A1*28 allele, respectively. However, our evaluation did not reveal any association between severe neutropenia and UGT1A1*28 genotypes (data shown in Table 1). We speculated that the null association might be due to the failure of recording lowest counts of neutrophils in patients with relatively poor compliance or inconvenience in seeking medical advice during their unhospitalization. In addition, anticipated neutropenia might also be covered up by preventive interventions such as granulocyte colony stimulating factor (G-CSF) treatments, which were even recommended by doctors concerned after completing the OR, odds ratio; CI, confidence interval; CRC, colorectal cancer; IRI, irinotecan; Beva, bevacizumab; Cape, capecitabine; C225, cetuximab; IFL, irinotecan and 5-fluorouracil; U, unknown; UFT, uracil/tegafur; OX, oxaliplatin; GC, gastric cancer; LC, lung cancer; EC, esophageal cancer. a: Among the seven studies, one study of gynecologic cancer took the regimen IRI + cisplatin with irinotecan dose of 60 mg/m 2 (d1, 8, 15) every four weeks. b: Weekly regimens were considered equal dosage as biweekly schedule when summarized in the table. c: One study of lung cancer took the regimen IRI, IRI + cisplatin or docetaxel with irinotecan dose of 50 to 60 mg/m 2 every four weeks. d: One study of Lung, stomach and colon cancer took the regimen IRI + platinum, IRI + others, FOLRIRI with irinotecan dose of 50 to 100 mg/ m 2 .

Reference year
Disease, stage

UGT1A Polymorphisms and Treatment Efficacy
Given the predictive value of UGT1A1 polymorphism in irinotecan-induced toxicity, genetic testing of these loci prior to treatment could tailor irinotecan therapy, enabling targeted surveillance and preventive measures in order to reduce the risk of chemotherapy-related side effects. Moreover, knowledge of which crowd will benefit (response and/or better survival rates) from the medical procedure could assist implementers in making an informed decision when prioritizing chemotherapy regimens. However, relatively less evidence supports the link between UGT1A1 genotype and irinotecan treatment efficacy, and the existing data are controversial.
It is reported that 35-55% of patients respond to irinotecan-based first-line chemotherapy, and the disease control rate is about 75%-85%, while both the rates are almost reduced by half when the regimen is taken as a second-line treatment [4][5][6][7] . UGT1A1*28 and UGT1A1*6 variants contribute to reduced UGT1A1 expression or decreased enzymatic activity 18,19 , and are linked to SN-38 glucuronidation, therefore, it is pharmacologically plausible that UGT1A1 genotype is connected with tumor response 37,38 .
A meta-analysis comprising fifteen Asian trials was conducted by Chen et al. 39 to investigate the relationship between UGT1A1*6 alleles and patient response to irinotecan-based chemotherapy. Most studies involved in the meta-analysis failed to find the relationship between UGT1A1*6 and therapeutic efficacy, possibly due to small sample sizes and mixed analysis of various therapy line (first-, second-and third-line). Poor statistic power might be partially eliminated by meta-analysis, nevertheless, no association was found between UGT1A1*6 allele and tumor response or survival, either in pooled analysis nor subgroup analysis (shown in Table 2). Hence, UGT1A1*6 polymorphism is less likely to be a predictor of irinotecan, especially in CRC patients, for tumor response and survival outcome based on these available studies.
Emerging data represented the predictive value of UGT1A1*28 allele in therapeutic efficacy of irinotecanbased chemotherapy 36 . Results from the meta-analysis by Liu et al. 40 revealed that UGT1A1*1/*28 or UGT1A1*28/*28 genotype was an unfavorable predictor for overall survival (OS) compared with wild-type genotype. Dias et al. 41 considered the evidence in Liu's meta-analysis was not strong enough to support the trend conclusion owing to their insufficient analyses of original data. A meta-analysis included 58 studies by Liu et al. 42 demonstrated an increased response rate in patients harboring UGT1A1*1/*28 or UGT1A1*28/*28 genotypes, but a null association between UGT1A1*28 and survival. Although most of the studies involved in these meta-analyses suggested a null association between UGT1A1*28 polymorphism and survival outcome, four studies showed predictive roles of UGT1A1*28 in irinotecan-treated patients, as a favorable indicator for progression-free survival (PFS) 14 or an unfavorable index for overall survival (OS) 15,16,43 . These inconsistencies may be partially attributed to diverse schedules of irinotecan, relatively small sample sizes, different study designs, and limited follow-up time.
A retrospective study focusing on gastric cancer treated with irinotecan as third-line therapy by Yamaguchi et al. 44 demonstrated a significant association between combined genotyping of UGT1A1*28 and *6 and OS outcome in univariate analysis, in which carrying variant allele is an unfavorable indicator for OS (hazard ratio [HR], 1.525; 95% CI, 1.033-2.251) compared with no carriers, but the significance faded away when adjusted in multivariate model (data shown in Table 2). If validated in prospective designed study, the combined indicator of UGT1A1*28 and *6 is promising in facilitating stratification of gastric patients for individualized third-line treatment options.
In our previous study 36 , which was prospectively designed to investigate the role of UGT1A1*28 polymorphism in therapeutic efficacy in Chinese metastatic CRC patients treated with irinotecan-based first-line chemotherapy, PFS and OS were co-primary end points, meanwhile, objective response rate (ORR) and disease control rate (DCR) were also evaluated. UGT1A1*28 carriers tended to have a reduced likelihood of objective response (ORR=22.7%) compared with the wild-type genotype (ORR=39.1%; OR, 0.444; 95% CI, 0.194-1.018; p=0.055). No significant difference was observed in groups divided by genotypes with respect to DCR. UGT1A1*28 variant genotype was predictive of worse PFS (median=7.5 months; HR, 1.803; 95% CI, 1.217-2.671) and OS (median=13.3 months; HR, 1.979; 95% CI, 1.267 to 3.091) compared with wild-type genotype (median PFS=9.8 months; median OS=20.8 months) (seen in Table 2). Since patients with UGT1A1*28 allele showed an unfavorable therapeutic response and susceptibility to irinotecan-induced toxicity (see Table 1), it is not recommended to carry out irinotecan-based regimen as first-line procedure in mCRC patients with UGT1A1*28 variant.
Opposite to the role in our study, UGT1A1*28 polymorphism seemed to be associated with increased clinical benefit and tumor response in the study by Toffoli et al. Patients bearing homozygous UGT1A1*28 were less likely to experiencing disease progression (OR, 0.19; 95% CI, 0.04-0.89), and had a significantly reduced risk of progression or stable disease compared with the wildtype genotype (OR, 0.32; 95% CI, 0.12-0.86). Analysis of time to progression (TTP) revealed a significant decrease in patients harboring UGT1A1*28/*28 (HR, 0.52; 95% CI, 0.31-0.90) and UGT1A1*1/*28 genotypes (HR, 0.73; 95% CI, 0.55 to 0.98) compared with the wild-type genotype. With respect to OS, no significant survival advantage was observed in UGT1A1*28 carriers. Data were shown in Table  2.

Meta-analysis
Chen et al. [38]  It is often considered that premature drug suspension and dose reduction as well as administration delays due to toxicity can decrease anti-tumor activity. We further investigated the connection between genotypes, survival outcome and dose reduction in the previous study [36] . UGT1A1*28 carriers tended to have an elevated likelihood of dose reduction compared with no carriers, although not statistically significant (OR, 2.156; 95% CI, 0.984-4.725; p=0.055). Dose reduction was significantly associated with decreased PFS (p<0.001) and represented a trend towards decreased OS (p=0.060). Therefore, dose reduction affected PFS, but whether it had an impact on OS needed further study. Additionally, subgroup analysis of patients treated without dose reduction showed that UGT1A1*28 allele was still an unfavorable predictor of PFS and OS. Since patients with UGT1A1*28 allele had more susceptibility to adverse effects (see Table 1) and less clinical benefits than wild-type genotype, it is not recommended to carry out irinotecan-based regimen as first-line procedure in mCRC patients with UGT1A1*28 variant.

Conclusion and Clinical Application of Potential Biomarkers
Genetic diversity exists among ethnics and individuals. UGT1A1*6 allele is frequently observed in Asian population, while rarely found in Caucasian population. UGT1A1*28 is an extremely common variant in Caucasian, and of a lower frequency in Asian. UGT1A1*28 and UGT1A1*6 (mainly in Asian) polymorphisms are promising predictors for irinotecan-induced toxicity in CRC patients receiving irinotecan-based chemotherapy. UGT1A1*28 variant is of some relevance to clinical advantage or disadvantage, but there are no sufficient evidence to support its role in therapeutic efficacy predicting.
Genetic testing could provide important insights for making individualized therapeutic strategies. In the USA, routine genotyping tests are performed typically for UGT1A1 *1/*1, *1/*28, and *28/*28 genotypes. It is recommended by the United States of America Food and Drug Administration (FDA) that when irinotecan is administered as a single-agent, a reduction in the starting dose by at least one level should be considered for patients with UGT1A1*28/*28 genotypes, and subsequent dose modifications should be made based on individual tolerance. However, the precise dose reduction in this crowd is not clear 21 . The Dutch Pharmacogenetics Working Group (DPWG) of the Royal Dutch Association for the Advancement of Pharmacy (KNMP) suggests starting with 70% of the standard dose for UGT1A1*28 homozygote carriers, and increasing following dose according to neutrophil counts if the patient tolerates the initial dose well 45 . In Asian populations, both UGT1A1*28 and *6 are taken into consideration when making irinotecan dose adjustment, and concurrence of *28 and *6, even when heterozygous, alters the disposition of irinotecan remarkably, potentially increasing susceptibility to toxicity 46,47 . An initial reduction of irinotecan is recommended for patients with UGT1A1 *6/*6, *6/*28, and *28/*28 genotypes in Japan 22 .
However, due to the lack of prospective data, it is yet unknown whether initial dose reduction leads to an altered antitumor effect, and whether the routine dose is sufficient for objects without variant homozygotes. The use of genetic testing for dose modification might be performed in selective cases: when the patient calls for aggressive treatment (e.g. shrinking the tumor for excision), genotyping might allow higher dosing for individuals with UGT1A1*1/*1 or *1/*28 genotypes 48-51 ; for patients prefer maximizing quality of life, genotyping might allow lower dosing for those harboring UGT1A1*28 homozygotes 15,23 .
To date, given the inconsistent result of the predictive effect of UGT1A1 on therapeutic efficacy, recommendations are given mainly based on the toxicity data of irinotecan and UGT1A1 genotypes. Furthermore, as 5-Fu is included in the combined chemotherapy protocol, dihydropyrimidine dehydrogenase deficiency (DPYD) polymorphism also needs to be considered in the predictive indicator. A few allelic variants of DPYD involved in the synthesis of nonfunctional or poorly functional enzymes expose patients to an increased risk of 5-FU-related adverse events (e.g. thrombocytopenia and stomatitis)35. Current genetic testing helps to identify patients with high risk of developing irinotecan-induced toxicity, and enables informed dosing, targeted surveillance and prophylactic measures. Further investigations are needed for building an optimal genetic prediction model with the potential to both reduce the burden of toxicity and improve survival.