| BackgroundGemcitabine is a nucleoside analogue which achieves its antitumor effect through the damage on cell replication. Gemcitabine-based chemotherapy regimenshave demonstrated activity in various solid tumors, including non-small cell lung cancer, breast cancer, pancreatic cancer, and sarcomas. However, it is characterized by a narrow therapeutic window. It is prone to induce adverse drug reactions such as myelotoxicity. Some studies have shown that two single nucleotide polymorphisms in the coding genes for cytidine deaminase (CDA):G208A and A79C might influence the plasma concentration of gemcitabine after administration, consequently effect on the adverse drug reaction and its efficacy in cancer patients. However, it has lead to controversial in Caucasians. And it has not been tested in Chinese populations.ObjectiveThe objectives of the current study are as follows.1. To establish a rapid, accurate and reliable method for genetic screening of common SNPs in CDA gene.2. To determine allele frequencies of CDA G208A and A79C in both Chinese healthy volunteers and cancer patients.3. To establish a method to determine the concentration of gemcitabine in human plasma.4. To study the influence of CDA A79C on plasma concentration and gemcitabine-associated adverse drug reactions in Chinese cancer patients.MethodsAn allele-specific polymorphism chain reaction method was established to determine the genotypes in CDA A79C site. Meanwhile, a reported restriction fragment length polymorphism method was adopted to determine the genotypes in CDA G208A site. Based on the establishment of those two genotyping methods, the allele frequencies at those two sites were determined in both Chinese healthy volunteers and Chinese cancer patients.An HPLC-MS/MS method was modified according to the previous reports and established under the experimental conditions in our lab to determine the concentration of gemcitabine in human plasma. Through the collection of blood samples at fixed time points after gemcitabine infusion and the follow-up toxicity monitoring data, the correlation between CDA polymorphism and gemcitabine plasma concentration, as well as gemcitabine-associated toxicity were established.ResultsThe minor allele frequencies of CDA A79C in Chinese healthy volunteers and in cancer patients were12.3%(n=102) and12.0%(n=50), respectively. These results were comparable to previous reports, showing that the allele-specific PCR method was accurate. It could be used to genotyping. For CDA G208A, the minor allele frequency in healthy volunteers was1.9%(n=52) and it was not detected in cancer patients. No differences were found between healthy volunteers and cancer patients for both SNPs. The minor allele frequencies of A79C and G208A in Chinese population were12.1%(n=152) and1.0%(n=102), respectively.The patients’peripheral bloods were collected at two time points:20minutes and90minutes, after the end of gemcitabine infusion. The plasma concentration of patients in the mutant group (those who carries CDA A79C mutant allele, genotypes A/C or C/C, n=9) at the20and90minutes’time points were3.03±1.70μg/ml and0.10±0.07μg/ml, respectively, while the average plasma concentrations in the wild-type group (genotype A/A) were2.39±1.14μg/ml and0.11±0.20μg/ml. No significant differences were found in the plasma concentrations between the two groups (p=0.191for the20minutes’time point and p=0.872for the90minutes’ time point).Myelosuppression was found to be the major gemcitabine-associated toxicity after follow-up toxicity monitoring for each patient for at least three cycles of treatment. The most frequent adverse drug reaction was leucopenia. It happened in81.1%of patients. After the analysis of patients according to genotype groups, the frequency of severe neutropenia in patients in the mutant group (62.5%,5/8) was significantly higher than that in patients in the wild-type group (17.2%,5/29)(p=0.021). Meanwhile, the frequencies of severe leucopenia and thrombopenia were25.0%(2/8) and12.5%(1/8). They were also higher than those in the wild-type group (13.8%,4/29for leucopenia and6.5%,2/29for thrombopenia). A tendency was observed that the patients in the mutant group were at a higher risk of developing severe adverse drug reactions, though it did not reach statistical significance.Conclusion The allele-specific PCR method for the genotyping of CDA A79C was successfully established in the current study. It is accurate and it could be used for further studies. The minor allele frequencies of CDA A79C and G208A in Chinese populations are12.1%and1.0%, respectively. Although no significant correlation was found between CDA A79C and plasma concentrations of gemcitabine after drug administration in patients, those who carry the mutant allele are at a higher risk of developing severe gemcitabine-associtated adverse drug reactions. |
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