Feasibility Research About PHLPP And MDK As MRD Target Gene And Establish The Monitoring System For MRD

MRD(minimal residual disease)refers to leukemic cells persisting within the patient after achievement of a complete release.These cells have the potential to form a regrowing leukemic population,which clinically occurs as relapse of leukemia.In fact,about 10¹²leukemic cells are present in a patient at diagnosis and are reduced by an induction therapy resulting in a CR,at this stage up to 10¹⁰leukemic cells may still persist.Due to the lack of sensitivity of the methods even large amounts of residual cells are not detectable by cytomorphology,.cytogenesis fluorescence in situ hybridization,flow cytometry,while RQ-PCR technology was widely used for its rapid,sensitive and simple,specific advantages.In addition,comparing to the traditional prognosis features,such as age,WBC,and chromosome mutation,the level of MRD comprehensive reflects pharmacokinetics,drug genetics and chemotherapy sensitivity of leukemic cells.Our object is to establish the monitoring system of broader coverage and more specific for residual leukemia by RQ-PCR technology,then can offer reference information for clinical treatment.Using PCR technology for MRD detection,the key is to look for specific gene signs of tumor;this mark can be a reliable representation of malignancies clone,and in the course of leukemia remained relatively stable.The fusion gene of chromosomal translocation is the ideal molecular markers for MRD detection.But only 40% patients specific fusion gene.are detectable in only 20-25%of all leukemia patients. The rearrangements of and TCR genes play important role in ALL patients,while Ig and TCR are specific for everyone and easy to transfor to another type.In order to search for coverage of leukemia MRD monitoring target gene,we select the medium-term factor gene(midkine,MDK)and PHLPP gene in current study of solid tumors as a target,based on clinical results,analysis the feasibility of PHLPP MDK gene as a target gene for MRD detection.MDK gene is the combination of heparin growth factor family members located in11 p11,through mitosis and cell proliferation to promote angiogenesis,induced malignant cell transformation,and anti-apoptotic function influencing tumor cell proliferation,differentiation and apoptosis,and the mRNA and protein expression of more tumors has increased.Many studies found MDK gene can induce tumor drug resistance through anti-aging gene,cell-mediated resistance mechanisms.At present the research for MDK limited to solid tumors,but leukemia research is less.Another new tumor suppressor gene PHLPP(PH domain leucine-rich repeat protein phosphatase)is a kind of protein kinase,which search from the 2 kinase PH structure domain of the human genome database,locating at the 18 q21.33,found by Newton et al in 2005.Research has shown that the gene is low expression in cancer,colorectal cancer cell lines,and in another study,Japan scholars have reported the gene in patients with acute myeloid leukemia and myelodysplastic syndrome patients with high expression,and low expression in normal and remission after chemotherapy.So it is necessary to test the PHLPP level in leukemia patients.MRD becomes increasing important in the risk-adapted management of patients with leukemia,our reseach sets up six fusion gene and WT1 gene detection methods,the seven genes are respectivly:â‘ BCR-ABL fusion gene forms from the long arm of chromosome 9 on the C-abl oncogene translocation to the long arm of chromosome 22 points on the fault zone bcr,and over 95%of the slow promyelocytic leukemia patients and 25%to 30%of adult patients with acute lymphoblastic leukemia cells in the blood may appear t(9,22)(q34,q11).â‘¡AML1-ETO fusion gene is due to chromosome t(8,21)(q22,q22)formed,and the accident rate is10% -12%,for the most is with AML-M2.The signs of the fusion gene have the better prognosis.â‘¢PML-RARA formed by the PML 15th gene on chromosome and the RARa 17th gene on chromosome(retinoic acid receptor genes),and AML-M3 is the specific fusion genes,account for 10-15%of new cases of the young people.â‘£CBFβ-MYH11 is a common fusion gene for acute myeloid leukemia(AML)M4Eo, by chromosomal abnormalities as Inv(16)(p13,q22)/t(16,16)(p13,q22),so this type of patients have good prognosis.⑤MLL-AF4 involves the most common genetic abnormality for 11 q23/MLL,chromosomal abnormalities for t(4,11)(q21,q23),and the incidence rate of acute leukemia is 5%-10%,having poor prognosis.â‘¥SIL-TAL is most common gene fusion for the T acute lymphoblastic leukemia(T-ALL),and chromosomal abnormalities is Del(1)(p32,p32).⑦WT1 gene(wilms tumor gene)is the most relevant with leukemia,isolating from wilms tumor cells in patients as a tumor suppressor gene,located 11 p13.WT1 gene frequently expresses in AML cells, participating in the process of leukemia,and the expression of the protein can maintain the viability of leukemia cells.The study shows that WT1 gene can be used as target genes of residual leukemia detection.RQ-PCR technology,useing of specific TaqMan probe,significantly improved the accuracy of PCR quantitative product and reduced the pollution of product operation,to truly achieve the quantitative.Sensitivity can reached 1×(10^-5-10^-6).We choose patients treated in our department from March 2007 to March 2008 as reseach object,including 126 AML patients,47 ALL patients,8 MDS patients,26 CML patients and 20 healthy blood donor.Conventional methods for extracting leukemia RNA reverse transcriptase cDNA for backup.Primers and probes were designed using Beacon Designer 7.0 design software based on their laocation on two separated exons. Firstly conduct residual leukemia target gene and gene GADPH by qualitative PCR, and amplified products of positive samples was extracted plasmid.And then measure several times to copies as the dilution to different gradients,making standard curve for fluorescence quantitative PCR.Using steward genes as the reference,based on standard curve,it measures different periods of target gene expression. Result:â‘ PHLPP gene in cells proliferation has higher expression and no related to leukemia diseases,not suitable as the target gene for MRD detection.â‘¡MDK gene in adult ALL and AML is the high expression,its high level have correlation with the subtype of leukemia,central nervous system leukemia,non-release after standard chemotherapy,while has no correlation with age,gender,relapse,WBC level when diagnose,is a sign of bad prognosis;MDK gene level which have correlation with乐leukemia processes can be as target gene of MRD detection;â‘¢97%initial treatment AML patients and 95%initial treatment ALL patients can find ideal target gene for MRD detection;â‘£initial treatment and relapse samples of target gene are overexpression,and after remission,the target sample of the gene expression level was significantly declining,comparing to the initial treatment and relapse,the results are statistically significant(P＜0.05);⑤MRD levels rise frequently is meaningful for recurrence,and MRD sustained low level has better prognosis.Conclusion:We test the level of MDKmRNA by RQ-PCR continuously in leukemia patients for the first time in and abroad,and proof that MDK is an ideal target for MRD.PHLPP gene in cells proliferation has higher expression and no related to leukemia diseases,not suitable as the target gene for MRD detection. Applying RQ-PCR technology to continuously monitor six fusion genes and two pan-sign genes,we have established the monitor system for broader coverage and more specific for MRD detection,for more important value of monitoring recurrence predict of disease by a variety of target genes.