The Study Of Monitoring In Imatinib-resistant Chronic Myeloid Leukemia Patients

Background and objective:Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the presence of the Philadelphia chromosome(Ph) which arised from a balanced translocation between chromosomes 9 and 22.This translocation results in the fusion of the c-ABL oncogene on chromosome 9 with the BCR gene on chromosome 22.The resulting BCR-ABL fusion gene encodes a protein named P210^BCR-ABL which is a constitutively active tyrosine kinase.By catalyzing the phosphorylation of the tyrosine residues in specific sites of P210^BCR-ABL itself and a host of substrates,P210^BCR-ABL deregulates multiple cytoplasmic and nuclear signal transduction pathways that result to the abnormality of proliferation,differentiation, attenuation and apoptosis. P210^BCR-ABL is not only critical in the pathogenesis of CML but also a potent target for treatment.Imatinib is a 2-Phenylaminopyramidine compound that is a potent inhibitior of P210^BCR-ABL and it mada a great breakthrough, for the treatment of CML .Five years after the 1106 patients has started treatment ,and with a median of 60 months of follow up in the IRIS trial,about 7% of the patients with chronic phase(CP) in the imatinib group had progressed to the accelerated phase(AP) or blast crisis(BC),the CML-relative mortality is 5%. Despite significant hematologic and cytogenetic responses,resistance occurs,particularly in patients with advanced disease. 432522The main mechanisms of resistance include point mutations of BCR-ABL tyrosine kinase, genomic amplification of BCR-ABL,over expression of BCR-ABL transcripts, overexpression of MDR-1 gene,α1-acid glycoprotein,the activation of other kinase.At present,two important aspects of imatinib resistance research are BCR-ABL dependent mechanisms and BCR-ABL independent mechanism.point mutations in the ABL kinase domain and the reactivation of P210^BCR-ABL is considered to be the most prevalent mechanism of resistance to Imatinib and can be found in about 30%⁹0% patients with resistance to imatinib.In 2006,the GIMEMA group analysised point mutations in 370 CML patients,and found 52% of AP,75% of myeloblatic crisis,and 83% of lymphoblastic patients can be found to have mutations.The number of reported mutations detected in patients treated with imatinib is approximately 70 described at 50 residues,Mutations can take place in ATP-binding loop(P-loop),activation loop(A-loop),catalysis domain and the contacting domain.The regions of the kinase domain where mutations are detected most commonly are the P-loop and at residues T315Iand M351 accounting for 60%-85%..Crystallographic analysis has revealed that mutations involving contact residues such as T315 confer resistance by impeding inhibitor access and/or eliminating critical hydrogen bonds.A second group of mutations,including those within the P-loop confer resistance by preventing abl from adopting the specific conformation required for high-affinity imatinib binding.Lastly,mutations in regulatory motifs such as the activation loop may stabilize an active conformation that is inaccessible to imatinib.Besides point mutations can change the biological properties of P210^BCR-ABL protein and its transforming potency.Mutations can not be detected even with high-sensetive technology in some patients with resistance to imatinib.For this part of patients,genomic amplification of BCR-ABL,over expression of BCR-ABL transcripts, overexpression of MDR-1 gene, and the activation of other kinase may be the underlying mechanisms.Over expression of BCR-ABL transcripts is also a important mechanism of resistance.The level of transcripts is increasing when disease relapses.Once imatinb therapy commences,frequent and regular monitoring of BCR-ABL level may identify poor responders and identify them at an early stage before disease progression and acquired resistance.Some of these patients may benefit from higher doses of imatinib and othe molecular targeted inhibitors.Besides,the log reduction in BCR-ABL,measured from the standardized baseline,was a good predictor of subsequent response and risk of progression.The achievement of major molecular response(MMR) by 12m,which means 3 log below the baseline of BCR-ABL level for untreated patients,was associated with probability of progression-free survival(PFS) and longer over survival. While failure to achieve a MMR is considered suboptimal and reassessment of therapy is recommended.In the recent year,with the universally application of Real-time quantatitive polymerase chain reaction(RQ-PCR) technology,which has the advantage of sensitive,quantatitive,prompt,monitoring of level of BCR-ABLmRNA became a indispensable sensitive indication of molecular response and minimal residual disease.Besides,a rising level of BCR-ABL is a trigger for kinase domain mutation analysis.The effective of imatinib therapy is closely associatede with the prognose.NCCN and the European leukemia NET suggest that multifaceted molecular monitoring shoule be conducted in CML patients during imatinib therapy,including regular bone marrow cytogenetic analysis,fluorescence in situ hybridization(FISH),RQ-PCR to monitor additional chromosomal abnormalities,gene amplification and increasing in BCR-ABL transcripts.NCCN also recommendes point mutation assay to patients in case of failure in imatinib,suboptimal response,hematologic or cytogenetic relapse,or rise in BCR-ABL transcripts.CRKL is an adaptor protein consisting of an src homology 2(SH2) domain and 2 tandem SH3 domain in the absence of any catalytic domain.CRKL is the major tyrosine-phosphorylated protein in the BCR-ABL transformed cells.This SH2-SH3 adaptor molecule CRKL could be an important mediator of the leukemogenic activity of BCR-ABL.CRKL involved in several important pathways induced by RAS/MARP,JAK/STAT,PI3K andβ1 intergin through link tyrosine kinase with downmstream effectors,and CRKL could form a stable complex with P210^BCR-ABL protein.In cells from patients with primary CML ,phosphorylation of CRKL(p-CRKL) occurs in cells as a direct consequence of BCR-ABL expression with the level of p-CRKL correlating well with the level of BCR-ABL protein.Importantly,p-CRKL is not detectable in BCR-ABL-negative cells.As a result,CRKL can be an surrogate to research the activity of P210^BCR-ABL.As we know,monitoring the activity P210^BCR-ABL protein is of great important for appraise the treatment during imatinib thrapy.The most direct measure of signaling through the BCR-ABL pathway is through the enzymatic activity of P210^BCR-ABL protein itself.Although readily measured in cell lines,this assay is difficult to perfume in a reproducible,quantatitive fasion with clinical material ,because BCR-ABL is subject to rapid degradation and dephosphrylation upon cell lysis.In the research for alternative measures of P210^BCR-ABL,wu found that the phosphorylation CRKL is a perfect alternative ,because of its specifically and constitutively phosphrylated by BCR-ABL.More important,the reduction of p-CRKL is a prognostic indicator and can be used as a predictor for cytogenetic and molecular response.In the study of Deborah W,patients with more than 50% inhibition of p-CRKL from baseline achieved high proportion of MMR compared the counterpart.The predictive power of in vitro imatinib sensitivity suggests that the actual level of BCR-ABL kinase inhibition achieved is important in determining response to imatinib,so CRKL an be used as a molecular monitor.In this study,we attempted to analysis abl kinase point mutations,the level of BCR-ABL transcripts and the phosphorylation level of CRKL of imatinib-sensitive and imatinib-resistant patients by nest RP-PCR,RQ-PCR and flow cytometry.,so as to explore the clinical meaning of molecular monitoring by the assayes mentioned above.Methods:1.Nested reverse transcriptase-polymerase chain reaction was performed on 39 bone marrow samples from 22 patients to amplify the ABL kinase domain,followed by direct sequencing and sequence homologous analysis.Afer 3 months or 6 months follow-up,point mutation analysis and FISH is conducted in patients with mutation and resistance to imatinib.2.To set up SYBR Green I PQ-PCR technology to detect and quantify BCR-ABL transcripts in imatinib-naive patients,imatinib-treated patients with negative results by FISH,and imatinib-resistant patientsm.Using formula: Folds= 2^{(-△△Ct)} ,we analysis the relative changes in BCR-ABLmRNA expression in imatinib-resistant patients and patients without BCR-ABL fusion signal by FISH to that of imatinib-naive patients. Independent- Samples T test was used to testify statistic meaning of BCR-ABL mRNA level between the two group.Bivariate test was done to analysis correlation between the results of RQ-PCR and FISH .3.Using FACS to study the phosphorylated CRKL protein of imatinib-naive CML patients,imatinib-sensitive patients and imatinib-resistant patients.The phosphorylation level is denoted by p-CRKL% and the mean fluorescence intensity (MFI).Using one-way ANOVA to identify the statistic meaning.By spearman Bivariate test to analysis the correlation of p-CRKL%,MFI and BCR-ABLmRNA inPart2.Results1 .Point mutations in the ABL tyrosine kinase domain and imatinib-resistantIn the total 15 imatinib-resistant patients analysed,The ABL domain point mutations was detected in 6(40.0%),resulting to 4 types of nucleotide changes:T315I(n=3),Y253H,E255K and F317L.Having been followed for 3-6 months,mutations can still be detected in those patients.A chimeric mutation F311I can be detected in patients NO5. This patient had a increasing in the proportion of BCR-ABL positive cell.Except for the patient with F317L who achieved CCyR, the proportion of Bcr-Abl of every patients with mutation is more than 90%.2.The level of BCR-ABL transcript and imatinib-resistantThe fusion gene transcripts level of imatinib-resistant patients is significantly higher than that of imatinib-treated patients with negative FISH results (2^{(△△CT)} is 1.50±0.49,0.12±0.20, respectively,P<0.05) .Theres is positive correlation between the level of BCR-ABL expression and the proportion of BCR-ABL cell analysised by FISH (r_s =0.933, P <0.05)3.The level of phosphorylated CRKL protein and imatinib resistantCRKL% and the MFI of p-CRKL between imatinib-naive CML patients, imatinib sensitive patients and imatinib-resistant patients is significant different(F=45.371, P <0.05; F=36.361, P<0.05) .There is a positive correlation between p-CRKL%, MFI and the expression level of BCR-ABL fusion gene (r =0.829, P<0.05; r=0.880, P<0.05) .Conclusions1 .Point mutations in the ABL tyrosine kinase domain is an important mechanism leading to imatinib resistant in CML patients. Analysis of point mutations is clinically useful for monitoring clinic status and efficay of the therapy.2.The expression level of BCR-ABL fusion gene by RQ-PCR can be used as molecular monitor to assess efficacy,identify disease relapse and aquired resistance.The rising of transcripts is also an important factor leading to resistant to imatinib.3. We assay the level of phosphorylated CRKL protein by flow cytometrytechnology to analysis the activity of P210^BCR-ABL tyrosine kinase protein,and set up a sensitive,reproducible,prompt technology to analyse the P210^BCR-ABL activity .The results of this part has a positive correlation with BCR-ABL expression level.4. The reduction of phosphorylated CRKL protein can be used to monitorresponse and predict prognose of imatinib. 10^BCR-ABL tyrosine kinase protein may still plays a role in imatinib resistance patients without points mutations.