【摘要】：目的:探討初治急性髓系白血病(acute myeloid leukemia,AML)患者誘導治療后獲得首次骨髓形態(tài)學完全緩解(complete remission,CR1)時,采用多參數流式細胞術(multiparameter flow cytometry,MFC)及Wilms瘤基因1(Wilms tumor1 gene,WT1)表達檢測微小殘留病(minimal residual disease,MRD),通過檢測MFC-LAIP聯(lián)合WT1的水平變化,對AML患者進行無復發(fā)生存(relapse-free survival,RFS)、總體生存率(overall survival,OS)等預后分析,以期指導臨床后續(xù)治療。方法:分析2010年10月至2016年10月山西醫(yī)科大學第二醫(yī)院血液科病房收治的持續(xù)接受治療的179例初治AML(M3除外)患者的臨床資料,所有患者均獲得CR/CRi/CRp,分析CR1時骨髓檢測MFC-LAIP、WT1水平在預測RFS、OS及指導個體化治療等方面的作用。1、骨髓(bone marrow,BM)標本來自AML患者。診斷及分類標準依據2016年世界衛(wèi)生組織(World Health Organization,WHO)。對所有患者初治及CR1后的BM樣本進行MFC-LAIP(leukemia associated immunophenotype)及WT1表達水平檢測。本實驗是在骨髓細胞水平進行的實驗。所用標本均來源于山西醫(yī)科大學第二醫(yī)院血液科因診療需要抽取的BM(獲得患者知情同意)。2、每例患者在初治及CR1后取BM 2-4ml,用FC500(Beckman公司)MFC檢測抗原表達及原始細胞計數,數據分析用CXP Analysis軟件。每個組合補償設定用未標記的同型對照。檢測前行Flow-check測試激光和補償的穩(wěn)定性。MFC進行免疫表型分析。單克隆抗體主要包括:CD45-PC7,CD38-FITC,CLL-1-PE,CD34-PC5,CD7-FITC,c MPO-PE,CD19-PC5,CD34-FITC,CD11b-PE,CD15-PC5,CD56-PE,CD117-PC5。同時采用實時定量熒光PCR(real-time quantitative polymerase chain reaction,RT-q PCR)檢測WT1。3、MFC分析以前向角/側向角(forward scatter/side scatter,FSC/SSC)射門去除死細胞和碎片,每管至少獲取10萬個細胞,以CD45和SSC(CD45/SSC)射門�；颊吖撬杓毎旧栃詾榭乖磉_超過20%的CD45/SSC細胞群,評估的最低有效細胞數是40�？乖磉_強度以陽性細胞群的平均熒光強度(mean fluorescence intensity,MFI)來確定,評估的最低有效細胞數是500。若在篩選時白血病細胞出現(xiàn)的區(qū)域內仍有細胞存在且表型異常,即判斷為殘留白血病細胞,以這些殘存的細胞占骨髓有核細胞總數的比例作為MRD的數值。MRD定量值≤10-4與為陰性,≥10-4之為陽性。4、WT1表達以歐洲白血病網絡公認的比值法計算,用RT-q PCR測定WT1和ABL基因,以ABL基因為內參。WT1/ABL×1000060為高表達,60為低表達。結果:1、179例初治AML患者中,男88例,女91例,中位年齡47歲(15-73)歲,依據WHO(2016)分型:AML伴t(8;21)29例,AML伴t(16;16)/inv(16)9例,AML伴inv(3)/t(3;3)1例,M0 5例,M1 12例,M2 15例,M4 53例,M4EO 4例,M5 25例,M6 4例,AML伴骨髓增生異常相關改變11例,治療相關AML11例。依據NCCN(2017)指南:低危37例,中危117例,高危25例。中位隨訪時間為23月(6-76)月,目前共100例患者復發(fā),復發(fā)多發(fā)生在CR1后2年內,76例早期復發(fā)(≤12個月)。死亡62例,其中55例死于復發(fā)。中位RFS 21月(4-75月),中位OS 28月(6-76月)。2、在73例AML患者中分析C型凝集素樣受體-1(C-type lectin-like receptor-1,CLL-1)在MRD檢測中的作用。研究發(fā)現(xiàn),CLL-1表達在正常對照骨髓細胞的粒細胞、單核細胞,不表達在淋巴細胞、有核紅細胞和CD34+CD38-細胞;在91.8%AML患者白血病細胞和粒細胞、單核細胞表達,在CD34+CD38-細胞表達陽性率為71.2%,且在疾病CR前后表達穩(wěn)定,而在CD34+與CD34-AML患者中表達無差異(P=0.43)。3、首次骨髓形態(tài)學CR后107例患者MFC-LAIP轉陰,72例患者MFC-LAIP仍為陽性。MFC-LAIP陽性患者與陰性患者一般特征相比,發(fā)病時外周血白細胞計數、BM、MFC及外周血原始細胞比例更高,血小板計數更低,多為合并中樞神經系統(tǒng)白血病(central nervous system leukemia,CNSL)、高白細胞計數、治療相關、中高危患者,初次誘導CR率低,但差異均無統(tǒng)計學意義(P值均0.05),MFC-LAIP陽性患者2個療程未CR比例明顯多于陰性患者(P=0.029),RFS、OS明顯縮短(24月vs 11月,35.5月vs 21月,P0.001)。4、73例AML患者中,57例(78.1%)患者初診WT1高表達,16例(21.9%)患者初診WT1表達不高,其中9例在CR1后WT1高表達,低、中、高危分別2例、4例、3例,老年患者4例,7例發(fā)生早期復發(fā)。WT1表達水平與外周血白細胞計數、血紅蛋白濃度、血小板計數、乳酸脫氫酶(lactate dehydrogenase,LDH)、β2微球蛋白(β2-microglobulin,β2-MG)、預后分層無相關關系。5、多因素分析發(fā)現(xiàn)MFC-LAIP、WT1水平為影響AML患者RFS、OS的獨立因素。分析不同危險度分層患者,發(fā)現(xiàn)MFC-LAIP水平為影響中危、高�；颊逺FS的獨立因素;中危患者OS的獨立影響因素,而不是低�；颊逺FS、OS的獨立影響因素。6、評估MFC-LAIP、WT1水平在AML患者預測復發(fā)、評估預后中的效能。發(fā)現(xiàn)MFC-LAIP≥1.35%組與1.35%組相比5年RFS率明顯降低(P0.001),該界值的靈敏度為0.575,特異度為0.818;MFC-LAIP≥1.70%組與1.70%組相比5年OS顯著降低(P0.001),靈敏度為0.714,特異度為0.769。CR1后WT1水平≥40患者與40患者相比18月RFS明顯降低(P=0.049),CR1后WT1水平≥50與50患者相比,20月的OS明顯降低(P=0.02);MFC聯(lián)合WT1表達檢測CR1后MRD可提高AML患者預測RFS、OS的靈敏度,而不影響特異度。進一步亞組分析不同療程達CR患者,發(fā)現(xiàn)仍可提高靈敏度,不影響特異度,但權重不同。結論:1、CR1后MFC-LAIP是影響AML患者RFS、OS的獨立因素,可能對CR后分層治療扮演重要甚至主導角色,但在不同危險度分層患者中權重不同。CLL-1可以作為AML診斷、MRD檢測及靶向治療的標記。2、CR1時WT1水平分別≥40及≥50預示更短的RFS、OS;初治時表達正常,CR1后WT1高表達者,多為中高危、老年患者,復發(fā)率高。3、MFC聯(lián)合WT1檢測可作為AML MRD檢測的良好手段,可提高MRD檢測水平的靈敏度,而不影響特異度。
[Abstract]:Objective: To investigate the first bone marrow morphologic complete remission (complete remission, CR1) after the first treatment of acute myeloid leukemia (AML), and to detect small residual disease by multiparameter flow cytometry (multiparameter flow cytometry, MFC) and Wilms tumor gene 1. Esidual disease, MRD), by detecting the level changes of MFC-LAIP combined with WT1, the prognosis analysis of AML patients without recurrent survival (relapse-free survival, RFS), overall survival rate (overall survival, OS), etc., in order to guide the clinical follow-up treatment. Methods: analysis from October 2010 to October 2016 in the Department of Hematology at the second hospital of Shanxi Medical University. The clinical data of 179 patients with AML (except M3) were treated with continuous treatment. All patients received CR/CRi/CRp. The analysis of CR1, MFC-LAIP, WT1 level in predicting RFS, OS, and individualized treatment, was used for.1. The bone marrow (bone marrow, BM) standard was derived from the patients. Diagnosis and classification criteria were based on World Health in 2016. Tissue (World Health Organization, WHO). MFC-LAIP (leukemia associated immunophenotype) and WT1 expression levels were detected for all patients and BM samples after initial treatment and CR1. This experiment was conducted at the level of bone marrow cells. All specimens were derived from the BM (obtained) from the hematological diagnosis and treatment of the second Hospital of Shanxi Medical University. The patient informed consent).2, each patient was taken BM 2-4ml after initial treatment and CR1, and FC500 (Beckman) MFC was used to detect antigen expression and primitive cell count, and CXP Analysis software was used for data analysis. The unlabeled same type control was set by each combination. The immunophenotype was detected by Flow-check test laser and the stability.MFC of compensation. CD45-PC7, CD38-FITC, CLL-1-PE, CD34-PC5, CD7-FITC, C MPO-PE, CD19-PC5, CD34-FITC, CD11b-PE, CD15-PC5, CD56-PE. Scatter, FSC/SSC) shot to remove dead cells and fragments. At least 100 thousand cells were obtained per tube, with CD45 and SSC (CD45/SSC) shot. The patient's bone marrow cells were stained positive for the CD45/SSC cell group over 20% of the antigen expression. The lowest number of effective cells evaluated was the average fluorescence intensity of the positive cell group with the 40. antigen expression intensity (mean fluorescence in). Tensity, MFI) to determine that the minimum number of effective cells evaluated is 500. if there are still cells and phenotypic abnormalities in the region of leukemic cells when screened, that is, residual leukemic cells are judged to be the proportion of the remaining cells to the total number of nucleated cells in the bone marrow as the number of.MRD quantitative values of MRD less than 10-4 and negative, or more than 10-4 The positive.4, WT1 expression was calculated by the ratio method recognized by the European leukemia network, and the WT1 and ABL genes were measured by RT-q PCR. The ABL gene was the high expression of the internal parameter.WT1/ABL x 1000060 and the 60 was low expression. Results: 1179 cases of early treatment AML, 88 male, 91 female, 47 years old (15-73), were classified according to WHO (2016): AML companion t (8; 21) 29 cases, 16; 1 6) /inv (16) 9 cases, AML with inv (3) /t (3; 3) 1 cases, M0 5 cases, M1 12 cases, M2 15 cases, M4 53, M4EO 4, M5 25 cases, M6 4 cases, AML companion myelodysplastic related cases. In the 2 years after CR1, 76 cases had early recurrence (less than 12 months). 62 cases died, of which 55 died of recurrence. Middle RFS 21 months (4-75 months), middle OS 28 months (6-76 months).2, and the role of C type lectin like receptor -1 (C-type lectin-like receptor-1, CLL-1) in MRD detection in 73 patients with AML. Granulocytes, monocytes, not expressed in lymphocytes, nucleated red cells and CD34+CD38- cells; in 91.8%AML patients, the expression of leukemic cells and granulocytes, mononuclear cells, expression in CD34+CD38- cells was 71.2%, and the expression was stable before and after the disease CR, and the P=0.43.3 and the first bone were expressed in the patients with CD34+ and CD34-AML. In 107 patients with CR, MFC-LAIP turned negative, and 72 patients with MFC-LAIP were still positive for positive.MFC-LAIP, compared with the negative patients. The proportion of peripheral blood leukocyte count, BM, MFC and peripheral blood primitive cells was higher, the platelet count was lower, and more of the central nervous system leukemia (central nervous system leukemi). A, CNSL), high white cell count, treatment related, high risk patients, the initial induction of CR rate was low, but the difference was not statistically significant (P 0.05), MFC-LAIP positive patients were not CR in 2 courses more than negative patients (P=0.029), RFS, OS significantly shortened (24 months vs November, 35.5 month vs 21 months, P0.001), 57 cases (78.1%) patients initially diagnosed 1 high expression, 16 cases (21.9%) patients with early diagnosis of WT1 expression was not high, of which 9 cases of high expression, low, middle, high risk 2 cases, 4 cases, 4 cases, 3 cases, 4 elderly patients, 7 cases of early recurrence.WT1 expression and peripheral blood white blood cell count, hemoglobin concentration, platelet count, lactate dehydrogenase (lactate dehydrogenase, LDH), beta 2 microglobulin (beta 2-) Microglobulin, beta 2-MG), prognostic stratification without correlation.5. Multifactor analysis found MFC-LAIP, WT1 level as an independent factor affecting RFS and OS in AML patients. Analysis of different risk levels of stratified patients showed that the level of MFC-LAIP was the independent factor in the risk of middle risk, the risk of RFS was independent; the independent factors of OS in the middle risk patients were not the low risk patients RFS. The independent influence factor.6, evaluate the MFC-LAIP, WT1 level in the AML patient to predict the recurrence and evaluate the efficacy in the prognosis. It was found that the RFS rate of MFC-LAIP 1.35% groups was significantly lower than that of the 1.35% group (P0.001), the sensitivity of the boundary value was 0.575, the specificity was 0.818, and the MFC-LAIP > 1.70% group was significantly lower than the 1.70% group (P0.001) and the sensitivity was 0.714, When the degree of specificity was 0.769.CR1, the level of WT1 was more than 40 in 18 months compared with that of 40 patients (P=0.049). The OS of 20 months was significantly lower than that of 50 patients after CR1 WT1 level (P=0.02), and the sensitivity of MFC combined WT1 expression was improved without affecting the specificity. The findings still improve the sensitivity without affecting the specificity, but the weight is different. Conclusion: 1. After CR1, MFC-LAIP is an independent factor affecting RFS and OS in AML patients. It may play an important and even dominant role in the stratified therapy after CR, but the weight different.CLL-1 in different risk stratification patients can be used as AML diagnosis, MRD detection and targeting therapy marker.2, At CR1, the level of WT1 is more than 40 and more than 50 indicates shorter RFS, OS; the expression is normal at the beginning of the treatment. The high expression of WT1 after CR1 is most high risk, the elderly patients, the recurrence rate is high.3. MFC combined WT1 detection can be used as a good means of AML MRD detection, which can improve the sensitivity of MRD detection level without affecting the specificity.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.71

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相關期刊論文 前1條

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本文編號：2175128