The Clinical Effect And Drug Resistence In Chronic Myeloid Leukemia Patients Treated With Imatinib

Background:Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by BCR-ABL fusion gene which is generated by the ABL gene on chromosome9with the BCR gene on chromosome22. General view is that allogeneic hematopoietic stem cell transplantation(allo-HSCT) is the best therapeutic method before1999and the transplant-related-mortality(TRM) and infect-related-mortality were obviously decrease for development of reduced intensity conditioning allogeneic hematopoietic stem cell transplantation and application of new antifungal and antiviral drug in the recent10years. There was a report that the5-year survival of CML patients was approximately84.2%. Most of the researchers viewed transplantation as curative therapy for CML. Imatinib (Gleevec, Novartis; formerly called STI571) is a relatively specific inhibitor of the BCR-ABL tyrosine kinase. Brian J et al. reported in an international randomized study of interferon and STI (IRIS) that the complete hematologic response (CHR), complete cytogenetic response(CCyR), and complete molecular responses (CMR)were96%,69%and85%respectively within12months and only7%CML patients progressed to accelerated-phase CML or blast crisis. The estimated overall survival of patients and event free survival who received imatinib as initial therapy was89%and83%at 60months. Other clinical trials abroad and domestic research about the analysis of Imatinib also have shown the better efficacy of complete hematologic response and complete cytogenetic response. So imatinib was rapidly adopted as a standard frontline therapy for CP-CML with those excellent results. Though the patients treated by imatinib have excellent results, there are some problems with imatinib. On the one hand, imatinib cannot eradicate leukemia stem cells completely, and there is no clinical trial proved that patients can stop therapy of imatinib. On the other hand, partly patients treated with imatinib will be acquired primary drug resistance or acquired drug resistance. The event of drug resistance was about18%in5-year follow-up, and the rate of patients in advanced treated by imatinib was considerably high, which the drug resistance was75%for patients in AP-CML, and95%for patients in BC-CML. So imatinib cannot cure CML for those facts. There are some reports declared that the combination of imatinib and allogeneic hematopoietic stem cell transplantation treatment for CML had highly rates of complete hematologic response and complete cytogenetic response. Some reports has indicated that the survival was superior for patients in the imatinib group compared to allo-HSCT. And other clinical researches had same conclusions,however, there is few reports about this two types of treatment for CML in our coutry. So, in the first part of our study, we would analysis the efficacy and survival of imatinib for CML patients compared to all-HSCT by collecting CML cases treated by imatinib or all-HSCT in our hospital in10years recently. Those dates could provide evidence of choices of treatment for CML, from which patients would benefit by the most optimal way to improve long-term survival.So far, there are approximately10%-25%CML patients who are resistant to imatinib treatment and ABL kinase point mutation is one of major resistance mechanisms. Mutations in the BCR-ABL kinase domain account for50%to90%of the imatinib resistance observed in patients. Structurally, the ABL kinase domain is made up of a highly conserved P-loop, catalytic domain, and activation loop. Some reports had claimed that the point sites characteristic of ABL kinase point mutation varies, which be resistant to imatinib in different degree, and ABL kinase point mutation had influence on the efficacy and long-term survival of CML patients. Therefore, the detection of ABL kinase point mutation for the patients resistant to imatinib is becoming more important. In the second parts of our study, we will discuss the relationship and clinical significance between ABL kinase point mutation and imatinib-resistance by gene fragment of ABL kinase mutation amplificated by PCR method with bone marrow or peripheral blood of CML patients.ASS gene that is fine amino acid biosynthesis genes is encoding the amino acid synthase fine, located in chromosome9q34.1, about200kb away from the ABL gene. And ASS gene is full length56kb,containing13-16coding exons and9%-28%CML patients of the deletion of chromosome9q34would delete ASS gene according to reports. Huntly etal had indicated that only48%patients with deletion of chromosome9q34who treated by IM could receive PCyR, which also indicated that there were relationship between deletion of chromosome9q34and efficacy of IM. Patients with deletion of chromosome9q34may have poor efficacy of IM, and other reports had also show that patients with deletion of ASS gene had poor prognosis. It is unclear the relationship between ASS gene and drug-resistance for CML patients. Therefore, in the third part of our study, we will discuss the relationship of ASS gene and drug-resistance and the clinical efficacy of CML patients with ASS gene.Objective:1. Compare the clinical efficacy between imatinib treatment and hematopoietic stem cell transplantation for CML-CP patients with stratified level.2. Discuss the relationship and clinical significance between ABL kinase point mutation and imatinib-resistance for CML patients.3. Discuss the relationship of ASS gene and drug-resistance and the clinical efficacy of CML patients with ASS gene.Methods:l.From February2002to February2012,198patients treated consecutively at the Nanfang Hospital, Southern Medical University were assigned to two groups according to treatment of imatinib or allo-HSCT. One hundred fifteen cases (male, n=77, female, n=38; median age,36yars old, rang,9-60yars old) in imatinib group were given imatinib at an initial dose of400mg daily and the dose was then adjusted according to the patient’s blood routine test and therapeutic response.. All the patients were evaluated for hematologic, cytogenetic and molecular response every3months. Eighty-three cases (male, n=48, female, n=35; median age,28yars old, rang,13-50yars old) in allo-HSCT group received myeloablative preconditioning regimen,and methotrexate (MTX) and cyclosporine A (CsA) which were used for graft-versus-host disease(GVHD) treatment and prophylaxis, partly combined with mycophenolate mofetil (MMF) and antihuman thymocyte globulin(ATG). The primary end points of the study were complete cytogenetic response (CCyR), relapse rate, overall survival (OS) and progression-free survival (PFS) after therapy.2. Nested reverse transcriptase-polym erase chain reaction was performed on samples from our hospital70patients to amplify the ABL kinase domain. Then, the amplified product was purified and sequenced in both direction. The homologous analysis was performed in combination of clinical data.3. FISH techniques, Bone marrow cell hybridization techniques and BCR/ABL1/ASS1probe were performed on BM cells from111patients in our hospital prepared according to conventional cytogenetic techniques. The hybridization and washing conditions were those recommended for the Vysis BCR/ABL1/ASS1detection system. The Tri-color DF FISH probes (Vysis, abbott) were applied and detected according to the manufacturer’s instructions. Combinations of biotinylated and Spectrum fluorescent-labeled probes were denatured with Cot1DNA, preannealed, hybridized, washed, and detected. All FISH images were captured and analyzed with a Smart Capture View Point multicolor imaging station (Digital Scientific, UK). For each marker, consistent data were obtained from a minimum of200Ph-positive metaphase cells in which the normal chromosomes9and22displayed normal signal patterns. The homologous analysis was performed in combination of clinical data.Results:1. In total,59(68.9%) patients treated over12months achieved a CCyR after12months in imatinib group, while67(95.7%) patients in allo-HSCT group. The relapse rates were14.8%(n=17) in imatinib group and10.8%(n=9) in allo-HSCT group (P=0.456). Ten-year cumulative OS rates were93.9%in imatinib group and77.1%in allo-HSCT group (P=0.015) and ten-year cumulative PFS rates of two groups were86.1%vs.88.0%(P=0.508). For Sokal rating stratified analysis, the ten-year OS rates of two groups were96.4%vs.68.0%(P=0.049) for high-risk patients,92.6%vs.57.1%(P=0.019) for intermediate-risk patients, while the ten-year PFS rates of two groups were89.3%vs.88.0%for high-risk patients (P=0.942),70.4%vs.85.7%for intermediate-risk patients (P=0.405).The ten-year OS rates and PFS rates were not significantly different for low-risk patients. The cumulative OS rates of two groups were94.7%vs.73.5%(P=0.019)for the patients who were not less than30years old, and the cumulative PFS rates of two groups were84.2%vs.94.1%respectively (P=0.147).2. The ABL domain point mutations were detected in32patients(45.7%) including16patients in chronic phase (CP),6patients in accelerated phase(AP) and10patients in blast phase(BP), which were detected as T315I, E255K,C475Y, Y253H,G321W, G250E, F317L, E258K, F359V, E459K, F311I. Sokal score with intermediate and high risk and Ph chromosome with complex karyotype were independent risk factors for ABL domain point mutations. The5-year overall survival(OS) was not significantly different for the patients with ABL domain point mutations and the patients without ABL domain point mutations (78.1%vs.84.2%, P=0.985), while the5-year cumulative event-free survival(EFS)of two groups was34.4%and68.4%(P=0.034), respectively. The rate of complete cytogenetic response (CCyR) was higher in patients treated with allogenic hematopetic stem cell transplantation (allo-HSCT) compared with patients merely treated with drugs(P=0.001).3.The detection of ASS gene deletion with BCR/ABL1/ASS1by FISH technique was simple,efficient and sensitive. And the results of ASS gene deletion were1G1RB1RGY and2G1RB1RGY signal mode. Among111CML patients,15cases (13.5%) were detected in the ASS gene deletion in our study.69patients who treated with IM over12months,4of11cases (36.4%) obtained CCyR after12months treaemnt in ASS gene deletion group, while34of58cases (58.7%) in ASS gene non-deletion group. There is no statistical singnificant difference between the two groups (36.4%vs.58.7%, P=0.202). Four of eleven patients occurred disease progression in ASS gene deletion group, while six of fifty-eigtht in ASS gene non-deletion group. There is statistical singnificant difference between the two groups (36.4%vs.10.3%, P=0.046).Conclusion:1.Imatinib confers significant survival and is superior to allogeneic hematopoietic stem cell transplantation for patients with chronic myeloid leukemia in chronic phase. Imatinib should be selected as a first-line therapy with few side effects and well tolerance.While allogeneic hematopoietic stem cell transplantation should be selected as a second-line therapy with good survival but with higher incidence of treatment-related mortality, GVHD and side effects.2. Detection of ABL domain point mutations in CML-CP patients resistant to imatinib was used for the adjustment for therapeutic options and improvement of prognosis. And allogeneic hematopoietic stem cell transplantation (allo-HSCT) was a more effective therapy for patients with advanced disease.3.The deletion of ASS gene of CML patients was13.5%in our study, and the BCR/ABL1/ASS1probe can effectively detect the deletion of ASS gene. The deletion of ASS gene may have influence on the imatinib treatment, accelerate disease progression and evaluate the prognosis of CML patients.