| Objective: As a chemotherapy drug ,mercaptopurine plays an important role in the treatment of childhood acute lymphoblastic leukemia (ALL) .Although the chemotherapy protocol is in improving, the importance of mercaptopurine has never been challenged. China Children's Leukemia Cooperative Group and the States"Eleventh Five-Year"Technology Support Program initiated a new program in childhood ALL-CCLG-08 in 2008. The program combined the most advanced technology,experience and philosophy is to set a standard, evidence-based, multi-center, randomized controlled clinical trial in China's ALL children. Mercaptopurine is applied with high dose of methotrexate (HDMTX) in the mM therapy, and in the maintenance treatment as a skeletal drug. The toxicity of mercaptopurine may affect the treatment efficacy and safty of the patients. TPMT is one of the key enzymes in the Mercaptopurine metabolic process. Serious toxicity may be resulted in when TPMT activity deficiency patients take standard doses of mercaptopurine. TPMT activity deficiency is closed associated with its alleles genetic polymorphisms. Many studied has already revaled that TPMT G238C, G460A, A719G single nucleotide polymorphisms(SNPs) are responsible for the toxicity in small part of the ALL patients with mercaptopurine. However, the SNPs allele types and frequency are different interms of the ethnic and geogaphy. Furthermore researchs found another mercaptopurine metabolic enzyme called ITPA on human chromosome 20,playing a crucial role.Excessive 6-me-ITP accumulation was found in the patients with ITPA activity deficiency , led to serious toxicity such as bone marrow suppression.ITPA activity deficiency is closely associated with its allele's genetic polymorphism. Previous studies has confirmed the SNPs of 94C> A site in exon 2 and the IVS2 +21 A> C site in intron 2 contributed to the decreased ITPA activity.This study is designed to observe the mercaptopurine-related toxicity in the childhood patients with ALL treated with CCLG-08 protocol and to investigate TPMT and ITPA gene polymorphisms in these patients. The relationship of the mercaptopurine related toxicity with the TPMT and ITPA gene polymorphisms will be then analized.Method: To observe the occurance of leukocytopenia, hepatotoxicity and pancreatitis in the patients received mM chemotherapy and the maintenane therapy. The genetic polymorphisms of TPMT G238C, G460A, A719G were determined by allele-specific PCR(ASPCR), PCR-restriction fragment length polymorphism(PCR-PFLP) and MALDI-TOF MS technique. The polymorphisms of ITPA 94C> A, IVS2 +21 A> C were determined by DNA sequencing. The relationships of TPMT, ITPA gene SNPs with mercaptopurine-related toxicity relationships were analized. Statistical analysis was done by software spss13.0.Results: 19.35% (n=24) of the ALL patients in the mM stage and 13.83% (n=13) in the maintenane therapy stage had experienced serious leukocytopenia. None of the patients had detectable hepatotoxicity in the mM stage, but 10.64% (n=10) of the patients had experienced hepatotoxicity in the maintenane therapy stage. No different occurance rate of mercaptopurine related toxicity has been found in the two groups of the maintenance therapy.The polymorphisms of TPMT G238C, G460A, A719G were detected in 149 ALL patients. Of them only one ALL patients were found with TPMT A719G heterozygous alleles. The study didn't find the relationship between the TPMT polymorphisms and the mercaptopurine related toxicity. (P>0.05). The total frequency of TPMT SNPs is 0.67%.The polymorphisms of ITPA 94C>A were detected in 149 ALL patients. The frequency of ITPA 94C>A site CA type is 32.89%(n=49) and the frequency of ITPA 94C>A site AA type is 3.36% (n=5). The total frequency of ITPA 94C>A SNPs is 36.24%. 20 ALL patients with ITPA 94C> A site CA/AA type had experienced serious leukocytopenia in the mM stage.11 ALL patients with ITPA 94C> A site CA/AA type had experienced serious leukocytopenia in the maintenance therapy stage. The ITPA 94C> A SNPs genetic polymorphism and the mercaptopurine-related hematological toxicity are significantly correlated(P<0.05). 2 ALL patients with ITPA 94C> A site CA/AA type had experienced hepatotoxicity in the maintenance therapy stage. The study didn't find the relationship between the ITPA 94C>A polymorphisms and the mercaptopurine-related hepatotoxicity(P>0.05).The polymorphisms of ITPA IVS2+21A>Cwere detected in 149 ALL patients. Of them none of the ALL patients were found with AC/AA alleles. The study didn't find the relationship between the ITPA IVS2+21A>C polymorphisms and the mercaptopurine-related toxicity(P>0.05).The total frequency of TPMT SNPs is 0%.Conclusion: Although with considerable ratio of the mercaptopurine related toxicity, the ALL patients in this study had been safely experienced the mM consolidation therapy and the maintenance therapy of CCLG-08 protocol.No different occurance rate of mercaptopurine related toxicity has been found in the two groups of the maintenance therapy of the CCLG-08 protocol. Continuous taking mercaptopurine didn't resulted in increased incidence of leucopenia comparing with continuous taking mercaptopurine 3 weeks and suspend 1 week.The mutation rate of TPMT gene G238C, G460A and A719G is 0.67% in the Han ethnic patients with ALL in Chongqing area. No relationship between TPMT G238C, G460A and A719G genetic polymorphisms and the mercaptopurine related leukocytopenia and hepatotoxicity were found in the study.The mutation rate of ITPA gene 94C>A is 36.24% in the Han ethnic patients with ALL in Chongqing area. A Significant correlation between ITPA 94C>A genetic polymorphisms and the mercaptopurine-related leukocytopenia was found . No relationship between ITPA 94C>A genetic polymorphisms and the mercaptopurine-related hepatotoxicity were found in the study. Detecting ITPA gene 94C> A genetic polymorphism before mercaptopurine treatment can predict mercaptopurine-related leukocytopenia.The mutation rate of ITPA gene IVS2+21A > C is 0% in the Han ethnic patients with ALL in Chongqing area. No relationship between ITPA gene IVS2+21A>C genetic polymorphisms and the mercaptopurine-related leukocytopenia and hepatotoxicity were found in the study. |
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