Clinical Observations on Associations Between the UGT1A1 Genotype and Severe Toxicity of Irinotecan

Irinotecan (CPT-11) is an S-phase-specific, semisynthetic derivative of camptothecin that interferes with DNA replication and cell division by inhibiting topoisomerase I (Fukuoka et al., 1992) and is now used widely, especially for colorectal and lung cancers (Negoro et al., 1991; Cunningham, et al., 1998; Kudoh et al., 1998; Rougier et al., 1998; Masuda et al., 1999; Uygun et al., 2013; Wei et al., 2013; Wu et al., 2013; Bozkurt et al., 2014; Zhang et al., 2014). Although it prolongs survival, patients who treated with chemotherapy include irinotecan is often accompanied by unpredictable and sever side effects. It causes severe diarrhea and neutropenia in 20% to 35% of patients treated (Negoro et al., 1991; Rothenberg et al., 1993; Rougier et al., 1998; Saltz et al., 2000; Othenberg et al., 2001; Vanhoefer et al., 2001; Fuchs et al., 2003). Fatal events (up to 5.3% prevalence) during single-agent irinotecan treatment have been reported (Vanhoefer et al., 2001). An innovative way of predicting the toxicity is strongly required. CPT-11 is metabolized to the active form, SN-38 which exerts its antitumor effect by carboxylesterase and is then

CPT-11 is metabolized to the active form, SN-38 which exerts its antitumor effect by carboxylesterase and is then 1 , Xin-En Huang 1 *, Xue-Yan Wu 1 , Jie Cao 1 1 1 , Jin Xiang 2 converted to the inactive form is by UGT (EC2.4.1.17) glucuronide is excreted in the small intestine via bile, where bacterial glucuronidase resolves the glucuronide into the former SN-38 and glucuronic acid (Takasuna et al., 1996). Interindividual differences in pharmacokinetics of SN-38 are suggested to cause the variation in drug effect (Gupta et al., 1994;Kudoh et al., 1995). Pharmacokinetic studies of irinotecan have reported large variations among individuals, as assessed by area under the concentration-time curves of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) and the inactive glucuronide metabolite (SN-38G), which is conjugated to UDP-glucuronosyltransferase (Gupta et al., 1994). UGT1A1 genotype affect the UGT enzyme activities and is believed to be the isoform primarily responsible for SN-38G formation, and interindividual variability in SN-38G formation is due to various UGT1A1 genotypes (Iyer et exons, which are alternatively spliced to four common exons, leading to mRNA isoforms, nine of which conduct to functionally active enzymes. In Asian, dinucleotide repeats in the atypical TATA-box region of the UGT1A1 promoter (UGT1A1*28 allele) have two sequences: (TA6, (TA)7, compose 3 genotypes: TA6/6 Wild-type UGT1A1*1/*1), TA6/7 Heterozygous (UGT1A1*1/*28) and TA7/7 Homozygous (UGT1A1*28/*28) An increased number of dinucleotide TATA-box region leads to a decreased rate of transcription initiation/expression of UGT1A1 (Beutler, 1998). Several studies suggest that patients homozygous for UGT1A1*28 are more likely to develop dose-dependent severe neutropenia and diarrhea compared with individuals with the no clinical characteristics factors contributing to increased toxicity risk using multivariable analyses reference genotype (*1/*1) (Beutler, 1998;Iyer, 2002;Innocenti et al., 2004;Marcuello et al., 2004;Carlini et al., 2005;Soepenberg et al., 2005;Toffoli et al., 2006;Ichikawa et al., 2008) Here we evaluated the association between UGT1A1 genetic variation and prevalence of severe toxicity with advanced lung and gastrointestinal tumors patients to and severe toxicity of CPT-11. In addition, this study also aimed to identify baseline clinical characteristics contributing to increased toxicity risk.

Patients
Patients were required to be pathologically/cytologically diagnosed with advanced lung and gastrointestinal tumors in Jiangsu Cancer Hospital and Research Institute from June 2006 to February 2014; to sign an informed consent before treatment; to have a score of karnofsky eligibility criteria included: adequate hematological (white blood cell count>3.0×10 9 and platelet count>150×10 9 ), liver (bilirubin and transaminases<1.5 times the upper normal limit) and renal function (creatinine leval<1.5 times the upper normal limit); patients were excluded from this study if they failed to complete two cycles of chemotherapy, or with any serious medical or psychiatric condition, or other malignancies. Pregnant or lactating women are also excluded from this study. We collect the patients' baseline clinical characteristics include sex, age, pathological reports, KPS, sites of metastasis, history of chemotherapy , serum total bilirubin levels before therapy and their UGT1A1 genotype. These factors were analyzed for importance for the side effects.
Patients were treated with CPT-11-based chemotherapy. The dosage and schedule of irinotecan administration that patients received were dertermined by their basic clinic characters and their UGT1A1 genotypes after they had the pharmacogenetic testing. The dosage adopted in our study is 100 mg/ 2 to 175mg/ 2 .We divide them into high-2 ) and low-dose grape (<125 mg/ 2 ). For lung cancer patients, CPT-11 was used with one Platinum include Cisplatin, Nedaplatin, Carboplatin or FOLFIRI regimen was used or CPT-11 was with Platinum, Fluorouracil , Pemetrexed or Raltitrexed . Modified FOLFIRI regimen was used every14 days, the others were every 21days. Because toxicity of irinotecan results in leukopenia and diarrhea is dose-limiting (Negoro et al., 1991), once patients occurred grade IV toxicity, the dosage would be adjusted next chemotherapy. No restriction was put on whatever the previous chemotherapy regimens course  in patients according to their basic clinic characters and their UGT1A1 genotypes. No standard was set to reduce the dose of second-course CPT-11 in patients who suffered severe diarrhea and neutropenia in their neutrophil levels recovered to the normal levels.
Toxicity was evaluated according to National Cancer Institute Common Terminology Criteria or Adverse Events version 3.0 criteria ( Table 1). The toxicity endpoints consisted of both GI and hematologic toxicities, and were analyzed separately. For GI toxicities, all patients were recored the incidence and severity of vomiting and diarrhea every day after irinotecan infusion. For hematologic toxicities, laboratory parameters were collected before, during, and after each cycle of chemotherapy. The most severe toxicities of every patient detectd were used for data as grade III-IV of toxicity. Once the toxicities occurred, the corresponding therapy would be implemented. For vomiting, antiemetics include 5-HT3 receptor antagonists and seroids were used. For diarrhea, Imodium was given: 4mg po once it happened, then 2mg po every 2 hours until the 12 hours after the last diarrhea. If time last over 48h, octreotide was given. For hematologic toxicities, G-CSF was for leucopenia or neutropenia, IL-11 for thrombocytopenia and ferrous succinate or EPO for anemia.
Genomic DNA was extracted with DNA extraction kit TCCCTGCTACCTTTGTGGAC-3', downstream: 5'-AGCAGGCCCAGGACaagt-3'.The PCR mix was 25ul: 2ul of 10×PCR buffer with 15mM MgCl 2 , 2ul of dNTP (2.5 mM), 1 ul of primer (10 um), 1ul of DNA Taq polymerase (5 U/ul), and 18.8ul of ddH 2 procedure was as follow: a initial denaturation step at If the PCR product hand is clear by electrophoresis, take 5ul of eligible specimen, mixed by adding 2ul SAP, We investigated the associations between side effects of CPT-11 and patients' UGT1A1 genotype characteristics included sex, age, pathological reports, KPS, sites of metastasis, history of chemotherapy and dosage used univariate analysis and multivariable logistic regression model. The relationship between serum total bilirubin levels and UGT1A1 genotype using rank test. And we slao Conduct the relationship between CPT-11-induced diarrhea and Leukopenia using correlation Analysis. SPSS version 16.0 software (SPSS Inc., USA) was used for all statistical analyses.

Results
89 patients were involved in this study with age between 18 and 80; male 68 and female 21; Lung cancer 24, Esophageal cancer 6, Gastric Cancer 9 and Colorectal at lest. The 6/6 variant of UGT1A1 was detected in 67 (75.3%) participants, the 6/7 variant was 22 (24.7%), while no 7/7 variant was detected. 9 (10.1%) patients      Table 2). The relationships between the side effects and their UGT1A1 genotypes or their baseline clinic characters were as followed. We also analyzed the factors of UGT1A1 genotype include serum total bilirubin and their baseline clinic characters.
The different UGT1A1 variants are showed in Figure  1 The univariate regression analysis of genotype of UGT1A1and baseline characters include age, gender, KPS score, history chemotherapy and metastasis number shows no statistical differences with p>0.05 (Table 3). It turns of UGT1A1. While the serum total bilirubins distribution of different UGT1A1 variants are showed in Figure 2. We The TA6/7 has a higher serum total bilirubin level than TA6/6 (p<0.036, Mann-Whitney U test; Table 4).
Because CPT-11 causes severe diarrhea and neutropenia in 20% to 35% of patients treated (Negoro et al., 1991;Rothenberg, et al., 1993;Rougier et al., 1998;Saltz et al., 2000;Rothenberg et al., 2001;Vanhoefer et al., 2001;Fuchs et al., 2003), and fatal events (up to 5.3% prevalence) during single-agent irinotecan treatment have been reported (Vanhoefer et al., 2001). We associated these two kind side effects with UGT1A1 genotypes, clinic characters and each other to detect the relationship between them by multivariate logistic analysis We took the sex, age, KPS, number of metastasis, history of chemotherapy, UGT1A1 genotype, dosage, leucopenia, neutropenia, primary cancer and pathological type into risk factors. By Multivariate logistic analysis, we can see that UGT1A1 genotype is the only factor with statistical difference, its OR is 6.108. It turns out that the risk rate to occur diarrhea of TA6/7 is 6.108 times of TA6/6, While sex, age, KPS, number of metastasis, history of chemotherapy, leucopenia, neutropenia, primary cancer with p>0.05 (Table 7).
For the neutropenia, we took the sex, age, KPS, number of metastasis, history of chemotherapy, UGT1A1 genotype, dosage, diarrhea, primary cancer and pathological type into risk factors. By multivariate logistic analysis, we don't p>0.05 (Table 8). It worth attention that the diarrhea may have some kind relationship with neutropenia (p=0.078). It means that the more severe the diarrhea occurs, the more severe neutropenia will be.

Discussion
CPT-11 is a broad-spectrum anticancer drugs with determinate effect that can prolong patients' survival.   But its usage always be limited by its severe side effects especially diarrhea and myelosuppression which are doselimiting toxicities (Negoro et al., 1991;Rothenberg et al., 1993;Rougier et al., 1998;Saltz et al., 2000;Rothenberg et al., 2001;Vanhoefer et al., 2001;Fuchs et al., 2003). Clinical administration dosage is generally calculated according to the body surface area or weight presently which is the group average dose (Reilly and Workman, 1993). but only a part of drugs calculated as above may get satisfactory effects and tolerable toxicity .The small changes in plasma concentration that affected by drug absorption, distribution, metabolism, and excretion may effects. Irinotecan therapy is also complicated by the pharmacokinetic parameter estimates (Gupta et al., 1994;1997) So it is very necessary that makes anti-tumor therapy entering the era of individualized treatment through the use of biological markers, drug metabolism genes, or pharmacokinetics detection, etc. UGT1A1 encodes the UGT which is essential for the inactivated of SN-38 that exert the antitumor effect of CPT-11. The UGT is also important for bilirubin metabolism. The abnormality in the promoter region of the gene has increased bilirubin and appears to be necessary for Gilbert's syndrome (Beutler, 1998). Our study shows that the serum total bilirubin of TA6/7 is higher than TA6/6.It is consistent with Bosma PJ' study.It makes sense that the higher serum total bilirubin UGT1A1*28. Also the distribution of UGT1A1*28 has a of UGT1A1*28 homozygote is not so high as compared to the Caucasian population (Araki et al., 2006). For Chinese, the TA6/6 of UGT1A1 is 79.6%, T6/7 is 18.2 % , and TA7/7 is only 2.2% (Ma Dong, et al., 2011). In our study, the distribution is consistent with the above. UGT1A1 is a important allele for avoiding the sever adversr events such as neutropenia and/or diarrhea caused by SN-38, the active form of CPT-11 (Ando et al., 2000). Pharmacogenetic testing of this allele is recommended prior to treat cancer patients with CPT-11 in US (Evaluation of Genomic Applications in Pratice and Prevention (EGAPP) Working Group. 2009). Our study shows that the usage of dosages adjusted by UGT1A1 genotype and patients' clinic charatcters caused the lower incidence of severe toxicity compared with 20%-30% showed in some studies (Negoro et al., 1991;Rothenberg et al., 1993;Rougier et al., 1998;Saltz et al., 2000;Rothenberg et al., 2001;Vanhoefer et al., 2001;Fuchs et al., 2003). It illustrates that pharmacogenetic testing of UGT1A1 is meaningful in clinic. In a Hoskins' metaanalysis of 821 cases in 9 clinic trials, severe toxicity was not increased in pediatric patients with the 7/7 genotype when treated with a low-dose (<150mg/m 2 ) protracted schedule of irinotecan, while mid-dosage in 150 ~ 250mg/ m 2 the dosage is around 100-175 mg/m 2 and it belong to the low-mid dosage in Hoskins' study, but UGT1A1 still make a statistical difference here. Our conlusion may make a Bias due to the reasons below: 1, The distribution of UGT1A1 is different between races, our study don't include 7/7 genotype. 2, Chemotherapy drugs Combined weren't included in hazard evaluation. 3, The treatment of toxicities is different now. It need more studies whether or not.
On the other hand, in our study, UGT1A1 is a independent risk factor for the increased CPT-11-induced diarrhea by univariate and Multivariate logistic analysis. There are many researches detecting the relationship and increased the risk of diarrhea (Innocenti et al., 2004;Raynal et al., 2010;Ma Dong et al., 2011). While some other suggest that UGT1A1*28 polymorphism is of some relevance to toxicity, however, it is less important than discussed in some trials (Rouits et al., 2004;Toffoli et al., 2006). But these studies all give a common conclusion: TA7/7 homozygous of UGT1A1 have a increased risk of and TA6/7 variations. Whatever in our study the risk rate of TA6/7 is 6.108 times of TA6/6 and should be detected to help guide the clinic use of CPT-11. While for neutropenia caused by CPT-11, we don't come out that UGT1A1*28 can increase the risk of it. Many studies abroad suggest that UGT1A1 genotypes are strongly associated with severe neutropenia, and could be used to identify cancer patients predisposed to the severe toxicity of irinotecan (Innocenti et al., 2004;Rouits et al., 2008;EGAPP Working Group, 2009). Some indicate that the risk of leucopenia or neutropenia in CPT-11-based chemotherapy is higher as the increase on dosage, especially in those who are administrated with medium or high irinotecan doses (>125mg/m 2 ) (Hu et al., 2010). While some others show that there is no relation ship between polymorphism of UGT1A1 the difference between TA6/6 and TA6/7 variations in severe neutropenia. Chemotherapy drugs combined and chemotherapy cycles are main factors interferencing our result. And more controlled studies with large patients Moreover, we observed that patients who occurred CPT-11-induced diarrhea always experience neutropenia mechanism to explain the association between them. But it worth a clinic note that when patients with CPT-11 occur diarrhea, we should test neutrophils more often to prevent the sever neutropenia happened and the more sever diarrhea is, the more sever neutropenia will be.
On Conclusion, Our study supports the result that UGT1A1*28 allele (s) will suffer an increased risk of sever irinotecan-induced diarrhea whether in mid-dosage or in low-dosage. While the UGT1A1*28 allele (s) doesn't increase the severe neutropenia. Higher serum total bilirubin is an indication that patients' t UGT1A1 genotype is not wide-type. And UGT1A1 genotype is