Examination of multiple UGT1A and DPYD polymorphisms has limited ability to predict the toxicity and efficacy of metastatic colorectal cancer treated with irinotecan-based chemotherapy: a retrospective analysis

Irinotecan is currently one of most important drugs in the management of metastatic colorectal cancer (mCRC) [1, 2]. Although the response rate and overall survival are greatly improved with the drug, about 30–50% of patients suffer severe toxicity, which particularly causes neutropenia and diarrhea [2]. UGT1A polymorphisms, especially UGT1A1*6 and UGT1A1*28, were previously noted to predict irinotecan-induced toxicity, but the results were inconstant [3, 4]. Based on a previous study performed in our center [5], UGT1A1*6 and UGT1A1*28 were found to be related solely to irinotecan-induced severe neutropenia, and not to diarrhea, as most studies in Asia indicated [4, 6]. The predictive sensitivity and specificity were relatively low, only 37.6% and 61.6%, respectively. Although the combined examination of multiple UGT1A loci improved the predictive sensitivity and specificity to irinotecan-induced toxicity, it still focused on predictability for severe neutropenia [7]. The results were based on studies with small samples. In actual clinical practice, severe diarrhea was more closely associated with mortality than neutropenia [8], but there is still no definite biomarker that can predict severe diarrhea in Asian patients [9, 10]. Moreover, irinotecan is commonly used in combination with fluorouracil, which also induces severe neutropenia and diarrhea. DPYD polymorphisms, which are associated with fluorouracil levels in vivo, are associated with the occurrence of fluorouracil-induced toxicity [11, 12]. In this way, it is necessary to find ways to improve the predictability of combined UGT1A with an examination of DPYD polymorphisms. This is the first large sample analysis of a combined examination of both UGT1A and DPYD polymorphisms to predict irinotecan-based chemotherapy-induced toxicity and the clinical response in Chinese patients.

This study was designed to evaluate the combinations of UGT1A and DPYD polymorphisms in predicting the occurrence of treatment-induced toxicity, clinical response and survival in China. Because of regional ethnic diversity, the genotype distribution differs in various parts of China. Based on the genotype frequency distribution in Chinese and other Asian patients from previous studies [13‐16], the genotypes of 661 patients have been examined at 9loci: UGT1A1*6, UGT1A1*27 (c.686C > A), UGT1A1*28, UGT1A7*2 (c.387 T > G), UGT1A7*3 (c.387 T > G, c.622 T > C), UGT1A7*4 (c.622 T > C), UGT1A9*22 (−118 T9 > T10), DPYD*5 (c.1627A > G), DPYD*2A (c.1905 + 1G > A), and DPYD c.1896 T > C. The relationship of each genotype to the risk of treatment-induced toxicities, response rate and overall survival are explored here. These findings may be used to establish a new panel, that would be more efficient in predicting treatment-induced toxicity or efficacy in China.

There were 661 mCRC patients who were finally enrolled in this study (all of the clinical data and the patients’ genotypes of UGT1A and DPYD are shown in the Additional file 1). Of the study population, 406 patients (61.4%) were male and 255 patients (38.6%) were female, and the median age was 56 years old (interquartile range [IQR] 47, 63). There were 98 patients (14.8%) who received irinotecan-based regimens as the first-line treatment and 563 patients (85.2%) who received irinotecan-based regimens as the second-line treatment or further. There were 71 patients (10.7%) who received single irinotecan-based chemotherapy and 590 patients (89.3%) who received irinotecan plus fluorouracil-based chemotherapy. All patients were eligible for toxicity evaluation and 634 patients were eligible for response evaluation. The incidence of severe diarrhea and neutropenia was 8.9% (n = 59) and 20.6% (n = 136), respectively. During the follow-up, 512 patients had disease progression and 346 patients were dead.

Among all of the patients, 49 of 71 patients who received single irinotecan-based chemotherapy had all of the UGT1A polymorphism loci examined, while 496 of 590 patients who received irinotecan plus fluorouracil-based chemotherapy had all of the UGT1A and DPYD polymorphism loci examined. The remaining 116 patients (including 22 patients who received irinotecan plus fluorouracil-based chemotherapy) only finished an examination of UGT1A1*6 and UGT1A1*28, because of examination failure and sample depletion. The genotypes are shown in Table 2.

In this cohort, the incidence of severe diarrhea and neutropenia was 8.9% and 20.8%. These were consistent with the previously reported results at the same center [5]. Clinical factors (including sex, age, primary tumor location, and chemotherapy regimens) did not show a significant relationship with treatment-induced severe diarrhea. Patients who received irinotecan-based chemotherapy as a second-line treatment or further had a lower risk of suffering severe neutropenia. The results are also shown in a previous report [19], which might be explained by more patients with better treatment tolerance receiving the second-line treatment or further. Female patients showed a potentially higher incidence of severe neutropenia, but with no statistical significance; however, in the report of Tsunedomi R et al., being female was an independent risk factor of severe neutropenia [7].

UGT1A genotype frequency and the effect on treatment-induced toxicity varied across ethnic groups. Early in 2005, UGT1A1*28 was recognized as a risk factor for irinotecan induced toxicities by the U.S Food and Drug Administration (FDA). In Asia, however, UGT1A1*28 were not applicable to the prediction of irinotecan-induced toxicity because of its low frequency. In this study, the genotype frequency of UGT1A1*6 and UGT1A1*28 were similar to previous reports in Asia [5, 20]. Both UGT1A1*6 and UGT1A1*28 related to G3–4 neutropenia, rather than delayed diarrhea, which was consistent with several large-sample analysis in Asia [4‐7]. Several small-sample analyses also noted that UGT1A1*28 and UGT1A1*6 could predict severe irinotecan-induced severe diarrhea [21, 22], which did not appear in the current study. A small sample analysis of Atasilp C et al., involving UGT1A1*6 and UGT1A1*28 were included in this analysis. Although individual UGT1A1*6 and UGT1A1*28 did not show a relationship with severe diarrhea neutropenia, the correlation of UGT1A1*6 and UGT1A1*28 revealed a significant association with severe neutropenia. Correlation of UGT1A1*6 and UGT1A1*28 also showed the same results in this study. In Thai patients, the UGT1A1*28 mutation frequency was nearly the same with Chinese patients (22.8% vs. 24.2%), while the UGT1A1*6 mutation frequency was lower than in Chinese patients (15.9% vs. 34.8%) [23]. The difference of polymorphism frequencies induced by ethnicity might explain the differences in the results of the two studies. UGT1A1*27 is a genotype only in Asians with lower UGT enzyme activity and low frequency [24]. Ten patients (1.8%) had UGT1A1*27 heterozygotes in this cohort, but only 2 patients suffered severe neutropenia. The incidence of severe toxicity was much lower than in previous reports [25]. The genotype frequency of UGT1A9*22 was 81.8% in this study, which was similar to findings reported in Japan. However, the UGT1A9*22 homozygotes in China were much rarer than in Japan (0.7% vs. 34.7%) [26]. UGT1A9*22 did not show an association with irinotecan-induced toxicity in this study. It has previously been reported that UGT1A9*22 variants have a lower risk of suffering irinotecan-induced severe neutropenia [7, 25, 26]. Chinese patients had similar UGT1A7*2/*3 frequency to Japanese patients, but a lower frequency than Greeks [26, 27]. Several studies have shown that UGT1A7*3 is associated with higher risk of suffering severe neutropenia [26‐29]. Tziotou M’s study also showed that UGT1A7*3 to be related to severe diarrhea [27]. In this study, UGT1A7*3 had a significant ability to predict severe neutropenia in univariate analysis, but the relationship did not appear to be significant in multivariate analysis. UGT1A7*3 was not an independent biomarker in the prediction of irinotecan-induced toxicity for Chinese patients. Among the patients who received targeted drugs, only UGT1A7*3 was found to be associated with a higher risk of G3–4 neutropenia incidence. Targeted drug treatment might affect the predictability of the toxicity of UGT1A polymorphisms. Regimens with different targeted drugs might also affect evaluation of toxicity. The influence of targeted drugs on the relationship between UGT1A polymorphisms and toxicity should be further studied. Finally, the results showed that UGT1A1*6 and UGT1A1*28 had an association with irinotecan-induced severe neutropenia. Patients with more mutant variants had a higher risk of suffering severe neutropenia; however, no other significant loci of UGT1A polymorphisms were found to set up a new panel to better indicate irinotecan-induced toxicity.

Fluorouracil is generally combined with irinotecan. DPYD polymorphisms influenced the activity of dihydropyrimidine dehydrogenase (DPD) considerably, which was associated with fluorouracil’s metabolism and ethnic variation also appeared in DPYD polymorphisms [14, 16, 30]. In western countries, it has been reported that DPYD*2A mutant variants contribute to a higher risk of severe toxicity [30]; however, DPYD*2A is rarely found. This was consistent with the findings of this study. Only 1 DPYD*2A heterozygote (0.2%) was found in this analysis. The ability of DPYD*2A to indicate fluorouracil-induced toxicity in China could not be assessed. DPYD*5 and DPYD c.1896 T > C had allele frequencies of 28.4% and 10.7%, respectively, which was consistent with previous reports [31]. In this cohort, it was noted that DPYD*5 associated with higher risk of severe diarrhea. However, in Zhang XP et al. and Yamauchi et al.’s study, DPYD*5 related to the incidence of severe neutropenia [31, 32]. In addition, the study of Felicia FS et al. showed that DPYD c.1896 T > C independently predicted severe fluorouracil-induced toxicity, which did not happen in this analysis [16]. A combined examination of UGT1A1*6, UGT1A1*28 and DPYD*5 was found to improve the predictive specificity for toxicity compared with an examination of UGT1A1*6 and UGT1A1*28 (53.1% vs. 85.6%) among patients receiving irinotecan plus fluorouracil-based chemotherapy.

The association between UGT1A and DPYD polymorphisms and clinical outcomes were analyzed, as well as the toxicity. The response rate and survival varied across different treatment lines. Among patients who received irinotecan-based chemotherapy as a first-line treatment, this analysis first noted that UGT1A1*27 contributed to worse OS than wild type variants, although the number of analyzed samples was small. Moreover, UGT1A1*28 contributed to a higher objective response rate, which was consistent with studies reported by Fujita and Toffoli G’s team [25, 33]. While, in Lu CY and colleagues’ study, UGT1A1*28 led to bad clinical outcomes [34]. This might be explained by a large number of factors affecting the survival. Single UGT1A gene polymorphisms were found to have only a limited ability to predict survival, and multiple chemotherapy regimens might also be involved. Unlike UGT1A, there have only been limited studies assessing the relationship between DPYD polymorphisms and survival. In this analysis, DPYD*5 mutant variants predicted better PFS in the first-line treatment of irinotecan plus fluorouracil-based regimens, and DPYD polymorphisms were not found to associate with overall survival.

Because this study was a retrospective analysis, bias was unavoidable. The value of this study relies on the large samples’ combined examination for UGT1A and DPYD polymorphisms. Although it was not possible to establish a new panel to improve the predictability of toxicity in this study, the analysis showed that more attention should be paid to homozygote of UGT1A1*6in the context of irinotecan-induced severe neutropenia, such as constantly monitoring the levels of neutrophile granulocytes and preventive treatment for neutropenia. For this reason, further studies should focus on polymorphisms of other genes related to irinotecan metabolism.

In brief, only UGT1A1*6 and UGT1A1*28 variants were associated with irinotecan-induced neutropenia, but not with diarrhea. A combined examination of UGT1A1*6, UGT1A1*28 and DPYD*5 were found to improve the predictive specificity of toxicity. UGT1A and DPYD polymorphisms were still limited to the prediction of clinical response. A combined examination of more UGT1A polymorphisms will not be helpful in improving predictive value of irinotecan-induced toxicity.