| Myelodysplastic syndrome (MDS) comprises a group of biologically and clinically heterogeneous clonal hematopoietic neoplasms characterized by aberrant myeloid differentiation, dysplastic changes, ineffective hematopoiesis and increased genomic instability, which clinically manifest as peripheral blood (PB) cytopenias and variably increased rates of leukemia progression. The disease might occur at any age, but more common in older adults, and 80% of whose ages is greater than 60. Foreign Epidemiology had shown that the annual incidence of MDS was 3 per 100,000 to 3.5 per 100,000, and the morbidity tended to increasewith age. MDS has male domiance of about 1.2:1, annual rates areestimated at 2.7 cases per 100,000 women, and 4.5 cases for every 100,000 men. It mainly afflicts people in elderly. The median patients age was 60-75 years old.There wasn no "gold standards" for diagnostics, and the dignosis of MDS synthesized multifactor, which should be made based on comperhensive information including a detailed detailed medical history, physical examination, blood routine, the level of serum ferritin,VB12, EPO, FA, bone-marrow smear, histopathology, FISH, flow cytometry, cytogenetics, gene mutations and other exclusive examinationse.Current the target of MDS treatment is to slove two problems:bone marrow failure and its complication, progression to acute myeloid leukemia. MDS are clinically heterogeneous disorders, its natural course and prognosis in various countries, thus, the choice of therapeutic schemes is depends on synthetic factor, for instance, IPSS-R (revised international prognostic scoring system), ECOG, age, patient-based compliance and so on. In general, low-intensity treatment is suitable for low-risk MDS, aim to promote haematogenous activity, enhance the quality of life and prolong survival time.And intense schedule suits high-risk cases inciouding Chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT), aim to achieve remission, improve natural progress, the prospects of survival and quality of life.The treatment efficacy of high-risk myelodysplastic syndrome is still in puzzle, and allo-HSCT remains the only option for cure. However, the diseases usually associated with the elderly,most of whom have lost the transplant opportunities. Moreover, for younger patients, various factors, such as the source of donor, cost, transplantation related complications, greatly restricte its applyment. Before the emergence of DNA hypomethylating agents (HMAs),there is no targeted medicine except for supportive care and hematopoietic growth factor, And for high-risk patients with MDS, the main treatment method is chemotherapy, but the reaction rate is very low with many adverse events, and cannot curb it progress to leukemia.DNA hypomethylating agents (HMAs), including Azacitidine (AZA) and Decitabine (DAC), may improve disease course, and the overall response of which was 40-60%..Azacitidine was approved by the Food andDrug Administration (FDA) for MDS in 2004 and decitabine in 2006. Now they are the only class of drugs and first line treatment, which are approved for the treatment of patients with higher-risk myelodysplastic syndromes (MDS). In addition, a plenty of randomized phase III and phase Ⅱ studies were later shown to confer an overall survival and efficacy benefit compared with supportive care and traditional treatment.And yet of all that, decitabine have taken advantages in MDS, there are still much dilemma. First, the cost is expensive which is all at their own expense in mainland China. The dosage and administration is 20mg/m2 for five days continuously, and the price is about 50,000 RMB per cycle. Second, the effct is slow. Several studies showed that treatment assessment should be evaluated after four to six cycles, which means the Chinese patients have to pay 200,000 to 300,000 RMB if they want to kown efficacy. Third, there is no withdrawal criterion.A sudden discontinuation in response patients lead to relapse or loss of efficacy in clinic, and the study in vitro confimed MHL1 gene remethylated when stopped to add decitabine. Forth, the incidence of drug resistance is high. A number of clinical trails have shown that decitabine provides significant clinical benefit for patients with MDS, but a number of patients do not initially respond (primary resistance), and a majority of patients lose response or progress despite continued therapy (secondary resistance). Additionly, in patients who do not respond to hypo-methylating therapy and those who relapse or progress after an initial response, prognosis is poor.Although clinical experience with these agents has outlined patterns for resistance and the failure of hypomethylating agents, there are no formal clinical studies that have established mechanisms underlying resistance to these drugs. Decitabine enters cells via the equilibrative nucleoside transporters hENTl and hENT2, which is similar to other cytosine nucleoside analogs (NAs). Once inside the cell, DAC is phosphorylated by deoxycytidine kinase (DCK) and becomes the monophosphorylated derivative 5-aza-dCMP. Subsequently,5-aza-dCMP is phosphorylated to generate its active form 5-aza-dCTP, which is incorporated into DNA, and it induces demethylation. Decitabine metabolites might also be substrates for catabolizing enzymes such as cytidine deaminase (CDA), which catalyze the inactivation of cytidine and deoxycytidine to uridine and deoxyuridine, thereby decreasing the amount of 5-aza-dCTP that can be formed.In vitro studies have indicated that DCK protein expression was low in the decitabine-resistant cell lines DU145 and Jurkat based on the DCK mRNA expression measured by real-time PCR, and the IC50 of decitabine was inversely correlated with DCK mRNA expression (R=0.63, P=0.038)[20]. In addition, a previous study revealed that decitabine could induce deficiency in deoxycytidine kinase (DCK) via different DCK gene mutations in vitro, which is related to their distinct interference with DNA replication Similarly, in an acute myeloid leukemia rat model, mutations in the deoxycytidine kinase (DCK) gene also induced resistance to 5-aza-deoxycyditine (DAC).In addition, recent studies showed that down-regulation of DCK specifically enhanced acquired resistance to another deoxycytidine analogue, gemcitabine, in pancreatic cancer. Given these data, we conclude that reduced DCK expression may cause secondary resistance, which may work through two pathways: DCK loss leading to a decrease in decitabine IC50, which reduces its intracellular drug concentration and insufficient levels of DCK for converting decitabine into its active form 5-aza-dCTP, meaning the drug is ineffective.The thesis is divided into three partsPart one:The clinical predictive factors of DNA hypomethylating agents in patients with MDSObjective:Hypomethylating agents (HMAs), including decitabine and azacitidine, have improved the outcome of patients with myelodysplastic syndrome (MDS). However, not all patients benefit from this therapy, and no reliable prognostic tool can predict the differential likelihood of benefit from HMAs, thus,we aimed to investigate the impact of clinical variables on response after decitabine or azacitidine treatment for MDS patientsMethods:Adults with a diagnosis of MDS who were referred to Guangdong General Hospital & Guangdong Academy of Medical Science were enrolled in this study according to FAB criteria. A total of 60 newly diagnosed patients with MDS received decitabine or azacitidine (3-23 cycles, median number of cycles:7) between November,2009 and November,2013.Results:1、 In this study, twenty patients received the approved schedule for AZA (75 mg/m2 for 7 days per cycle), and fourty patients received DAC (mostly 20 mg/m2 for 5 days per cycle). The median age of the 46 patients studied was 59 years, and the median number of cycles was 7 (range:3-23). The WHO diagnoses were RAEB-Ⅰ, RAEB-Ⅱ, and low blast count (20-30%) AML for 16,33, and 1 case, respectively. According to the International Prognosis Scoring System (IPSS), the cytogenetic risk was good for 38, intermediate for 10 and poor for 12 cases. According to MDS International Working Group (IWG) response criteria, the overall response rate for HMAs was 55%, and the baseline characteristics are shown in Table 1. There was no significant difference in response by treatment regimen (P=0.234). In addition, sex (P=0.759), age (P=0.069), IPSS risk group (P=0.115) and cytogenetic abnormalities (P=0.839) were not associated with response rate to HMAs. The only clinical feature significantly associated with response rate was disease status at diagnosis(P=0.033), driven largely by the high response rate of RAEB-Ⅰ and RAEB-Ⅱ patients2、 The overall response was 55%, and patients whose platelet count doubled after 2 cycles of treatment showed a significant higher response rate (P=0.004).With a follow-up of more than 24 months, there was a statistically-insignificant trend for OS prolongation in patients who achieved platelet count doubling compared to their counterparts (24 VS 16 months, P=0.273), especially for those treated with decitabine (38 VS 14 months, P=0.025). Multivariate analysis confirmed PLT doubling was an independent predictor for OS (P=0.021).Conclusion:Based on the results of the study, a two-fold or larger increase in platelet count after 2 cycles of HMAs suggested higher overall response and longer OS.Part two:The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndromeObjective:Decitabine is approved for the treatment of MDS, but resistance to this agent is common, and we want to determine the mechanisms underlying decitabine resistance, and investigate whether the hENT1, DCK, CDA and BCL2L10 gene expression level of genes could predict the response to decitabine for MDS patients.Methods:Adults with a diagnosis of MDS who were referred to Guangdong General Hospital & Guangdong Academy of Medical Science were enrolled in this study according to FAB criteria.28 newly diagnosed patients with MDS who received decitabine more than 2 cycles of standard treatment (2-23 cycles, median number cycles:1). A total of 14 patients had an overall response after treatment.14 patents showed no response. Among the responders,5 patients achieved more than 8 courses of mCR (continued mCR). but 5 patients relapsed or lost response despite continued therapy.In addition,3 relapsed AML patients after treatment with decitabine were also included in this study. A total of 44 BM and PB samples were collected in this study.28 samples were from 28 MDS patients(14 responders and 14 non-responders) at diagnosis.5 samples were from 5 MDS patients with continued mCR.5 samples were from 5 MDS patients at relapse.6 samples were from 3 AML patients at diagnosis and relapse.Results:1、we measured the mRNA expression of metabolism (hENT1, DCK, CDA) and apoptosis (BCL2L10) genes and found that the hENT1 mRNA level in the non-response group was significantly lower than that in the response group for patients with MDS (P=0.004). Similarly, we further examined the expression level of DCK, CDA and BCL2L10 and found that they did not affect the response rate (PDCK=0.687; PCDA=0.201;PBCL2L10=0.201).2、DCK, CDA, hENT1 and BCL2L10 expression level of males and females were 96.70±36.3 vs 128.72±36.3 (P=0.157),2048.69±1254.57 vs 1679.65± 1555.03 (P=0.295),191.91±66.68 vs 217.75±94.38 (P=0.295) and 1.19± 1.21 VS 1.45±0.75 (P=0.176) respectively. The results showed that gender does not influence all of the genes related to metabolism.3、 Furthermore, the DCK level was significantly reduced for relapse and other genes including CDA (P=0.484), hENT1 (P=0.208) and BCL2L10 (P=0.093) were not changed significantly. In addition, we further compared the mRNA expression level of DCK in bone marrow or peripheral blood from patients with mCR at diagnosis and continued mCR, and the results suggest that for continued mCR patients, the mRNA expression level of DCK did not decrease significantly (P= 0.222).The above-mentioned results demonstrate that the DCK expression level is a potential predictive indicator for secondary resistance in MDS and AML patients who received decitabine.Conclusion:These findings indicate that the decitabine metabolic pathway affects its therapeutic effects, lower hENT1 expression may induce primary resistance and down-regulated DCK expression may be related to secondary resistance.Part three:The relationship of ASXL1, TET2, TP53 gene mutations and response of HMAs and survival time in patients with myelodysplastic syndrome Objective:We want to determine the mechanisms underlying decitabine response, and investigate whether the ASXL1, TET2, TP53 gene mutations could predict the response and survival time to HMAs for MDS patients.Methods:In this study,53 adults with a diagnosis of MDS who were referred to Guangdong General Hospital & Guangdong Academy of Medical Science were enrolled. A total of 53 BM or PB samples were collected from these MDS patients before treatment, and we sequenced the ASXL1, TET2, TP53 gene mutations. Thus, we determined the relationship of ASXL1, TET2, TP53 gene mutations and response of HMAs and survival time in patients with myelodysplastic syndromeResults:1、In this study, according to MDS International Working Group (IWG) response criteria,29 patients responsed,and the overall response was 53.7%. Fourteen patients had TET2 gene mutation, and its frequency rate was 26.41%. Nine patients had ASXL1 gene mutation, and its frequency rate was 16.98%. Seven patients had ASXL1 gene mutation, and its frequency rate was 13.20%. Expect for one homozygous mutation, the remain others were heterozygous mutation, including point mutation, missense mutation, nonsense mutation, frame-shift mutation, insertion mutation and so on. Three patients contained both TET2 gene mutation and ASXL1 gene mutation, and one patient contained all the 3 gene mutations.2、In the fourteen patients with TET2 mutations,7 patients responded,and 7 cases did not respond, and TET2 mutation did not affect the response rate(P=0.620). Howover, in the nine patients with ASXL1 mutations,7 patients didn’t respond, and statistics analysis showed that the MDS patients with ASXL1 mutations responded poor (P=0.075), but it is necessary c to exlarge the samples’ quantity for further evidence. Moreover. TP53 gene mutations didn’t predict the response rate yet, but it was correlated with decreased survival (P=0.007), rather than TET2 gene mutation (P=0.513) and ASXL1 gene mutation (P=0.110).Conclusion:1、 These findings indicate that the MDS patients with ASXL I mutations responded poor.2、TP53 gene mutations was correlated with decreased survival in patients with MDS. |
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