| Background and Objective:It has been reported that95%Chronic myeloid leukemia patients (CML) have Ph chromosome. Now days molecular targeting therapy has became an important treatment option for clinical doctors. Imatinib, as a targeting inhibitor of the leukemia-initiating Bcr-Abl tyrosine kinase, has become a standard therapeutic agent for newly diagnosed chronic phase-CML (CP-CML) patients. The8-year IRIS trial demonstrated an event-free survival (EFS) of81%and an overall survival (OS) of85%in CML. Althongh imatinib has been successfully applied in CML management, many attention has been paid to its drug-resistant speciality. According to the best-cumulative hematological and cytogenetic response rates obtained from the IRIS trial and the response criteria of2009ELN. approximately10to25%patients were considered to be imatinib-resistant, which is the failare of achieving a complete hematological response (CHR) by3months or a complete cytogenetic response (CCR) by18months whith the therapy. Recent studies suggested that a wariety of factors could alter the intracellular concen-trations of the compound may recult in affecting its efficacy and subsequently modify its drug resistance. It has been shown that imatinib is a substrate for the P-glycoprotein (Pgp) efflux pump. However several experiment results raising the suspicion that Pgp expression itself may not be a clinically relevant predictor of IM resistance in population studies. However, protein expression levels may not be correlated with functionality. Dulucq and his collea-gues have evaluated polymorphisms in the multidrug resistant sequence and revealed a differential MMR rate to imatinib in three different single-nucleotide polymorphisms, suggesting that different sequences may alter the efficacy of efflux pump and result in differential responses to the IM therapy. Recently, uptake transporters, especially the organic cation transporter hOCT1, has been identified as a potential source of imatinib resistance. In clinic, different individuals may have different respond to the same drug, Some cases severe side effects or could effects drug-resistance. The heredity is a deterministic factor that can make different individuals with diverse drug metabolism and drug response. These individual diversity is attributed to the genetic polymorphism of genes codong which code drug metabolism enzyme, acceptor and drug transport protein. The methods used to analyze gene polymorphism include polymerase chain reaction-Restriction Fragment Length Polymorphism, single strand conformation polymorphism, polymerase chain reaction-denaturing gradient gel electrophoresis, Southern blot, High Performance Liquid Chromatography and polymerase chain reaction-sequence specific primer. In this study, we treated CML patients with imatinib and evaluated the efficacy. We studied the mRNA expression level of hOCT1(human organic cation transporter1) and ABCB1(adenosine triphosphate-binding cassette subfamily B member1) of bone marrow cells obtained from CML patients. For the patients with high expression level of ABCB1, we furthered analyzed C1236T、C3435T、G2677T single nucleotide polymorphism to study their function in modulating the efficacy of DM in CML trentment imatinib on the treatment Chronic myelocytic leukemia patients.Methods:1. mRNA expression of hOCTl and ABCB1on efficacy of Imatinib mesylate in chronic myelocytic leukemia1.1Study subjects:90cases of outpatient or inpatient CML patients in the hematology department of Nanfang hospital from August2008to February2011were studied. There were61male cases and29female cases. Median age was40(11-77) years old. Including88cases in chronic phase and two cases in accelerated phase. Imatinib was given at a median dose of400(300-800) mg orally per day and the median course of treatment was30(1-117) months. The samples were divided into three groups:drug-resistant group (17cases18.9%,12cases were primary drug resistant and5of them were secondary drug resistant), PCyR group (11cases,12.2%) and CCR group (62cases,68.9%).The expression level of hOCTl and ABCB1and their relationships to IM effectiveness were analzed in each group.1.2. Primer design and synthesis:we designed the primers of hOCTl, ABCB1gene and the housekeeping genes GAPDH in accordance with the GeneBank sequence.1.3. Total RNA were extracted from BM transcripted mononuclear cells of CML patients; and was reverse transcriptd into cDNA, The expression level of hOCT1and ABCB1were determined by fluorescence quantitative PCR. The specificity of PCR product the melting curve. An average of two Ct values were need to calculate the2-ΔCT value, which was considered as the specimen relative mRNA expression levels, ΔCT=(CT value of target gene-CT value of reference gene). The expression of hOCT1and ABCB1from drug-resistant group, PCyR group,CCR group and their relationships with different Imatinib treatment course were compared. Patients were divided into high expression group and low expression group according to the expression level of median levels of ABCB1mRNA in this study. And the time to achieve CCR in each group and proportionality were studied.1.4. All statistical analyses were performed with the SPSS13software(SPSS Inc, Chicago, IL, USA). Mann-Whitney U test was used to analyze in the expression of each gene in different each group. Correlations between variables were assessed by the Spearman rank correlation. Kaplan-Meier estimation was used to set up the survival curves. Two-sided analysis with P values<0.05were considered statistically significant.2. Influence of ABCB1gene single nucleotide polymorphism on efficacy of Imatinib mesylate in chronic myelocytic leukemia2.1. Study subjects:There were48cases of outpatient or inpatient CML patients in hematology department of Nanfang hospital from August2008to February2011were studied, and ABCB1mRNA high expression during we prophase study. There were32male cases and16female cases. Median age was38(11-68) years old. There were46cases in chronic phase and two cases in accelerated phase. Imatinib was received at a median dose400(300-800) mg orally per day and median course of treatment27(1-105) months. The samples were divided into three groups:no-CCR group, CCR group and MMR group.analyze the relationships to expression level of hOCT1and ABCB1.2.2To analyze the single nucleotide polymorphism of ABCB1gene. We have been detected C1236T、C3435T using polymerase chain reaction-Restriction Fragment Length Polymorphism and G2677T using polymerase chain reaction-sequence specific primer. Reaction product depuration were combined with agarose electrophoresis to determine the purpose gene segment. 2.3HWE software was used to set up the Hardy-weinberg test in the genotype frequency of the detected patients. Pearsonx2test was used to analyze the difference of Imatinib therapeutic effect in different genotype and allele frequency. All statistical analyses were performed with the SPSS13.Results:1. Analysis of purity and quality of RNA and cDNA:total RNA and cDNA were detected by UV Spectrophotometer, and the ratio were between1.8and2.0.2. The same cDNA sample was diluted by2-fold, The result was that amplification efficiency of the housekeeper gene and target genes were identical at the concentration range. Melting curve was done after amplification. It showed sharp single peak and no other specific peak with each gene. So we considered PCR products were homogeneous, no other non-specific amplification and primer dimmer. We could see that a single strip, no primer dimer and non-specific amplification by agarose gel electrophoresis. As to ABCB1we used the same principle.3. The hOCT1gene mRNA expression of CCR group was higher than drug-resistant group and PCyR group (P=0.008). The ABCB1gene mRNA expression of drug-resistant group was higher than CCR group and PCyR group (P=0.014).4. hOCT1and ABCB1mRNA expression were no significant difference between the five groups (3-6、-12、-24、-48、>48month) that divided by IM treatment course(P=0.270and0.367).5. The hOCT1high and ABCB1low expression groups were earlier than low and high expression groups in the time of patients first achieve CCR separately (P=0.006and0.049). During18month the percentage of achieve CCR patients in low expression groups of both genes were higher than high groups (97%Vs89%,97%Vs88%), especially in6month (76%Vs53%,74%Vs53%). The two genes mRNA expression had indirect correlation (r=-0.655, P<0.001) by using Spearman rank correlation analysis. 6. Genotype frequency Hardy-weinberg test, there were no different between actual number and theory (P>0.5). The crowd fited the Hardy-weinberg enetic balance, and suited to make population genetics analysis.7. There were no different in no-CCR group, CCR group and MMR group in ABCB1mRNA high expression level patients of ABCB1C1236T、C3435T、G2677T locus genotype and allele frequency. In no-CCR group C1236T T allele was higher than C allele(x2=4.00; P=0.046), C3435T、G2677T, C and G allele were higher than T allele(x2=7.111、5.444; P=0.008、0.02)Conclusion:1. The extracted RNA, cDNA are of high purity, good quality and can be used for the follow-up experiment.2. Primer designed for each gene is specific. And its amplification efficiency is consistent with housekeeper gene.3. The hOCT1gene mRNA expression of CCR group was higher than that of drug-resistant group and PCyR group. The ABCB1gene mRNA expression of drug-resistant group was higher than that of CCR group and PCyR group. Drug-resistant group accompany with hOCTl low expression or ABCB1high expression demonstrated that drug transport protein was an important factor in Imatinib therapy effect.4. Course of Imatinib treatment was correlated to the expression of the two genes, which indicated that hOCT1and ABCB1have reliable stability during the imatinib treatment, and have no relationship with the time of therapy.5. The hOCTl high and ABCB1low expression groups were earlier than low and high expression groups in the time of patients who first achieved CCR separately, the expression level of the two genes can affect the drug curative time.6. The ABCB1C1236、C3435T locus T and C allele associated primary failure or inadequate response of imatinib. In G2677T locus patients harboring a T allele have better progernosis than those with a G allele. |
PDF Full Text Request