楊波，蔡力力，汪海濤，朱宏麗，遲小華，于睿莉，楊洋，冉海紅，辛麗君，姚善謙，盧學(xué)春

急性髓細(xì)胞白血病(acute myelocytic leukemia，AML)發(fā)病率隨年齡增加而升高，發(fā)病中位年齡超過65歲，是老年人最常見的惡性血液病類型之一[1]。在絕大部分老年AML中，由于衰老相關(guān)的器官功能退化、合并基礎(chǔ)疾病多，患者往往不能耐受標(biāo)準(zhǔn)劑量化療。近來研究顯示，去甲基化療藥物地西他濱對(duì)骨髓增生異常綜合征(myelodysplastic syndrome，MDS)及其轉(zhuǎn)化的AML安全有效[2-3]，但常規(guī)劑量的地西他濱對(duì)老年患者的骨髓毒性作用使其應(yīng)用受到很大限制。對(duì)于老年AML是否存在最適的地西他濱治療劑量，即既能起到治療作用又不至于引起嚴(yán)重骨髓抑制，值得進(jìn)一步研究。我們的前期臨床研究發(fā)現(xiàn)，自體細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(cytokineinduced killer，CIK)能使部分高齡AML患者獲得血液學(xué)改善甚至是部分緩解[4-7]?？紤]到地西他濱通過表觀遺傳學(xué)調(diào)控治療白血病的潛在作用及CIK細(xì)胞相對(duì)特異性靶向殺傷白血病細(xì)胞的作用[8]，我們系統(tǒng)觀察了超低劑量地西他濱聯(lián)合自體CIK細(xì)胞治療1例80歲以上高齡MDS轉(zhuǎn)化AML的安全性及療效，現(xiàn)報(bào)道如下。

1 資料與方法

1.1 臨床資料 患者，男，83歲，因“乏力、盜汗3個(gè)月余”于2006年4月就診，既往有右肺肺泡癌、冠心病、慢性腎功能不全等病史(表1)。無特殊家族史。查體無明顯異常。血常規(guī)：白細(xì)胞(WBC)3.1×109/L，中性粒細(xì)胞(N)34%，淋巴細(xì)胞(L)56%，單核細(xì)胞(M)5%，血紅蛋白(Hb)143g/L，血小板(PLT)149×109/L。紅細(xì)胞沉降率、血清葉酸、VitB12含量正常。骨髓穿刺見紅系發(fā)育異常，占12%。鐵染色未見環(huán)形鐵粒幼細(xì)胞，診斷為骨髓增生異常綜合征–難治性貧血(MDS-RA)?；颊邚?008年8月開始出現(xiàn)貧血及血小板減少，間斷給予氨磷汀聯(lián)合重組人紅細(xì)胞生成素β治療，監(jiān)測血常規(guī)，WBC波動(dòng)于2.0～3.1×109/L，PLT 70～90×109/L，Hb 90～110g/L。2009年12月15日血常規(guī)提示W(wǎng)BC 7.06×109/L，M 16%；外周血涂片發(fā)現(xiàn)幼稚細(xì)胞(圖1A)；骨髄穿刺提示原始細(xì)胞占10.4%，幼稚單核細(xì)胞占2.4%，成熟單核細(xì)胞占11.2%(圖1B)；骨髓單個(gè)核細(xì)胞多重巢式PCR發(fā)現(xiàn)FLT3點(diǎn)突變，HOX11、C-kit、c-myc、GATA2等基因表達(dá)中度增高；染色體核型為(46，XY)，診斷為慢性粒單細(xì)胞白血病(chronic myelocytic mononuclear leukemia，CMML)，仍給予氨磷汀聯(lián)合紅細(xì)胞生成素治療。此后，患者外周血單核細(xì)胞計(jì)數(shù)及幼稚細(xì)胞比例逐漸增高，2010年2月20日WBC升高至17.62×109/L，其中原始細(xì)胞3%，單核細(xì)胞31%，骨髓穿刺示原始粒細(xì)胞占15%，幼稚單核細(xì)胞占23.6%，診斷為急性髓細(xì)胞白血病M4型(AML-M4)(圖1C)。

表1 本例既往疾病史Tab.1Retrospective history of the case

圖1 患者外周血涂片及骨髓象Fig.1Peripheral-blood smear and bone marrow hemogram from case

1.2 治療方案 確診AML-M4后即開始給予地西他濱(江蘇正大天晴制藥有限公司)聯(lián)合自體CIK細(xì)胞輸注治療，具體用法：地西他濱10mg d1-5，CIK細(xì)胞輸注(每次2×109～8×109)d14，rhIL-22mU d15-19，每周期28d(圖2)。自體CIK細(xì)胞的制備及回輸：給予胸腺五肽注射液20mg/d肌內(nèi)注射，連續(xù)1周。第8天晨起空腹采集外周靜脈血54ml，在我院基礎(chǔ)所免疫室(符合GMP條件)分離單個(gè)核細(xì)胞(peripheral blood mononuclear cells，PBMC)，用無血清培養(yǎng)基調(diào)整細(xì)胞濃度，加入rhIFN-γ使終濃度達(dá)到2000U/ml。將細(xì)胞懸液置于透氣性培養(yǎng)袋中，37℃、5%CO2條件下懸浮培養(yǎng)，次日加rhIL-21000U/ml，anti-CD3McAb 50ng/ml，培養(yǎng)第4、7、10、13天進(jìn)行細(xì)胞表型分析、調(diào)整細(xì)胞濃度、補(bǔ)充IL-2，使回輸時(shí)的CIK細(xì)胞表型符合如下標(biāo)準(zhǔn)：CD3+細(xì)胞比例大于70%，CD8+細(xì)胞比例大于40%，CD3+CD56+細(xì)胞比例不低于30%。培養(yǎng)第14天通過靜脈將CIK細(xì)胞回輸給患者。

圖2 超低劑量地西他濱聯(lián)合自體CIK細(xì)胞治療方案Fig.2Therapeutic regimen of decitabine combined with autologous CIK cells transfusion

2 結(jié) 果

患者共接受8個(gè)周期的治療?；熎陂g出現(xiàn)Ⅰ/Ⅱ度骨髓抑制及Ⅰ度胃腸道反應(yīng)。第2、3、8療程患者出現(xiàn)高白細(xì)胞血癥，最高達(dá)141.95×109/L，其中原幼細(xì)胞49%，單核細(xì)胞37%，中性粒細(xì)胞8%，淋巴細(xì)胞5%。加用依托泊苷(50mg d1-3)治療，高白細(xì)胞血癥得到控制。在間斷輸血治療下，患者Hb波動(dòng)于77～138g/L。血小板在第2個(gè)療程開始上升，第6個(gè)療程達(dá)到204×109/L(圖3)。骨髓象示原始、早幼粒細(xì)胞及幼單核細(xì)胞占16%。療效評(píng)價(jià)達(dá)部分緩解(圖1D)。2011年12月25日患者死于嚴(yán)重肺部感染引起的多臟器功能衰竭。自診斷AML至死亡總生存22個(gè)月。

3 討 論

老年AML具有自身的臨床生物學(xué)特點(diǎn)：①臟器功能退化，合并多種慢性基礎(chǔ)病，對(duì)化療耐受差[9]；②多有前驅(qū)血液系統(tǒng)疾病(常見為MDS)，造血處于衰竭狀態(tài)，化療后血象恢復(fù)慢；③染色體異常比例高[10]，耐藥率高，緩解后容易復(fù)發(fā)[11]。因此，老年AML治療難度大，預(yù)后差[12]。據(jù)文獻(xiàn)報(bào)道，80歲以上AML患者中位生存期僅6周[13]。Kantarjian等[13]分析了998例老年高危MDS/AML患者的臨床資料，共篩選出7個(gè)與總生存相關(guān)的預(yù)后指標(biāo)，其中，大于3個(gè)指標(biāo)的患者中位總生存期只有1個(gè)月。根據(jù)此預(yù)后評(píng)價(jià)標(biāo)準(zhǔn)，本例預(yù)期生存期為1個(gè)月(表2)。

圖3 患者病情演變過程及治療期間的血象變化Fig.3Hemogram change during progression and treatment

表2 本例生存相關(guān)危險(xiǎn)因素Tab.2Risk factors related to the predicting survival of the case[13]

老年AML的治療至今仍無標(biāo)準(zhǔn)方案，小劑量阿糖胞苷和最佳支持治療均不能延長患者的總生存[3,14]，長期生存(5年)率僅5%～15%[3,15-16]，因此，老年AML仍被歸為難治性白血病類型。故有必要針對(duì)老年AML開展臨床試驗(yàn)研究，探索高效低毒的治療方法，有效提高緩解率，延長生存率，改善患者生活質(zhì)量。

地西他濱是一種DNA甲基化轉(zhuǎn)移酶抑制劑，具有去甲基化作用，可重新激活由于DNA過度甲基化而失活的基因，從而誘導(dǎo)腫瘤細(xì)胞分化、凋亡[17-18]。多項(xiàng)臨床試驗(yàn)表明，小劑量地西他濱對(duì)老年MDS/AML具有一定療效，能夠提高生存質(zhì)量，改善血液學(xué)指標(biāo)，延緩MDS向AML的轉(zhuǎn)化，延長無進(jìn)展生存和總生存期[19-21]。綜合文獻(xiàn)報(bào)道，地西他濱治療60歲以上老年AML的單次劑量在15～45mg不等，一個(gè)療程的總劑量在100～200mg，治療總反應(yīng)率25%～64%，中位生存期5.5～12.8個(gè)月，治療相關(guān)死亡率13%～25%[15,22-23](表3)。但對(duì)于由MDS轉(zhuǎn)化而來的80歲以上高齡AML患者，當(dāng)前報(bào)道的地西他濱劑量由于血液學(xué)毒性大而難以適用，緩解后的維持治療也不能單純依靠地西他濱。本研究應(yīng)用超低劑量地西他濱聯(lián)合自體CIK細(xì)胞輸注方案治療MDS轉(zhuǎn)化的高齡AML，效果良好。本例診斷AML時(shí)87歲，合并右肺肺泡癌，病程中出現(xiàn)高白細(xì)胞血癥，預(yù)期生存僅1個(gè)月[13]，給予超低劑量地西他濱聯(lián)合自體CIK細(xì)胞治療8個(gè)周期，患者生存期達(dá)22個(gè)月，顯著高于文獻(xiàn)報(bào)道。對(duì)于本方案的療效機(jī)制，我們推測可能與地西他濱和CIK細(xì)胞的協(xié)同作用有關(guān)。地西他濱在高劑量時(shí)主要顯示細(xì)胞毒作用，而在低劑量時(shí)則表現(xiàn)為對(duì)腫瘤相關(guān)基因的表觀調(diào)控作用[24-26]，例如能使失活的抑癌基因重新表達(dá)，使腫瘤細(xì)胞表達(dá)新的癌抗原，從而增強(qiáng)CIK細(xì)胞對(duì)腫瘤細(xì)胞的識(shí)別和殺傷；反過來，CIK細(xì)胞具有潛在的增強(qiáng)體內(nèi)其他免疫效應(yīng)細(xì)胞功能的作用[7,27-29]，這也會(huì)提高患者的免疫力，進(jìn)而降低地西他濱治療后骨髓抑制期發(fā)生感染的風(fēng)險(xiǎn)。

表3 地西他濱治療老年AML用藥方案及療效總結(jié)Tab.3Dosage regimen and efficacy of decitabine in treatment of AML in elderly patients (≥60) with AML

總之，對(duì)于老年MDS轉(zhuǎn)化的AML，超低劑量地西他濱聯(lián)合CIK細(xì)胞輸注是一種可供選擇的安全有效的治療方法，未來尚需積累更多病例加以驗(yàn)證。

[1]Estey E. Acute myeloid leukemia and myelodysplastic syndromes in older patients[J]. J Clin Oncol, 2007, 25(14): 1908-1915.

[2]Lübbert M, Suciu S, Baila L, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediateor high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group[J]. J Clin Oncol, 2011, 29(15): 1987-1996.

[3]Lübbert M, Rüter BH, Claus R, et al. A multicenter phase II trial of decitabine as first-line treatment for older patients with acute myeloid leukemia judged unfit for induction chemotherapy[J].Haematologica, 2012, 97(3): 393-401.

[4]Yang B, Lu XC, Yu RL, et al. Repeated transfusions of autologous cytokine-induced killer cells for treatment of haematological malignancies in elderly patients: a pilot clinical trial[J]. Hematol Oncol, 2012, 30(3): 115-122.

[5]Liu Y, Bao EN, Yang B, et al. Clinical study of autologous cytokine induced killer cell infusion treating for elderly patients,with myelodysplastic syndrome[J]. J Exp Hematol, 2011, 19(3):787-792.[劉洋, 包爾寧, 楊波, 等. 自體CIK細(xì)胞輸注治療老年骨髓增生異常綜合征的臨床研究[J]. 中國實(shí)驗(yàn)血液學(xué)雜志, 2011, 19(3): 787-792.]

[6]Wang Y, Bo J, Dai HR, et al. CIK cells from recurrent or refractory AML patients can be efficiently expanded in vitro and used for reduction of leukemic blasts in vivo[J]. Exp Hematol,2013, 41(3): 241-252.

[7]Lu XC, Yang B, Zhu HL, et al. Cytokine-induced killer plus IL-2for elderly patients with a hematological malignancy[J]. Med J Chin PLA, 2010, 35(10): 1270-1272. [盧學(xué)春, 楊波, 朱宏麗, 等. 自體細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞聯(lián)合IL-2治療老年人血液系統(tǒng)惡性腫瘤的臨床經(jīng)驗(yàn)探討[J]. 解放軍醫(yī)學(xué)雜志,2010, 35(10): 1270-1272.]

[8]Pievani A, Borleri G, Pende D, et al. Dual-functional capability of CD3+CD56+CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity[J]. Blood, 2011,118(12): 3301-3310.

[9]Pedersen-Bjergaard J, Ersb ll J, S rensen HM, et al. Risk of acute nonlymphocytic leukemia and preleukemia in patients treated with cyclophosphamide for non-Hodgkin's lymphomas.Comparison with results obtained in patients treated for Hodgkin's disease and ovarian carcinoma with other alkylating agents[J]. Ann Intern Med, 1985, 103(2): 195-200.

[10]Pagano L, Mele L, Casorelli I, et al. Acute lymphoblastic leukemia in the elderly. A twelve-year retrospective, single center study[J]. Haematologica, 2000, 85(12): 1327-1329.

[11]Leith CP, Kopecky KJ, Godwin J, et al. Acute myeloid leukemia in the elderly: assessment of multidrug resistance (MDR1) and cytogenetics distinguishes biologic subgroups with remarkably distinct responses to standard chemotherapy. A Southwest Oncology Group study[J]. Blood, 1997, 89(9): 3323-3329.

[12]Li TT, Sun XD, Dong Z, et al. Comparison of molecular biology,immunological characteristics and clinical efficacy in patients with acute myelogenous leukemia with or without FLT3-ITD gene mutation[J]. Med J Chin PLA, 2013, 38(6): 501-505.[李婷婷, 孫雪冬, 董征, 等. FLT3-ITD陽性與陰性急性髓細(xì)胞白血病的實(shí)驗(yàn)室特征及預(yù)后比較[J]. 解放軍醫(yī)學(xué)雜志, 2013, 38(6): 501-505.]

[13]Kantarjian H, O'brien S, Cortes J, et al. Results of intensive chemotherapy in 998patients age 65years or older with acute myeloid leukemia or high-risk myelodysplastic syndrome:predictive prognostic models for outcome[J]. Cancer, 2006,106(5): 1090-1098.

[14]Miller KB, Kim K, Morrison FS, et al. The evaluation of low-dose cytarabine in the treatment of myelodysplastic syndromes: a phase-III intergroup study[J]. Ann Hematol, 1992, 65(4): 162-168.

[15]Büchner T, Berdel WE, Haferlach C, et al. Age-related risk profile and chemotherapy dose response in acute myeloid leukemia:a study by the German Acute Myeloid Leukemia Cooperative Group[J]. J Clin Oncol, 2009, 27(1): 61-69.

[16]Ziogas DC, Voulgarelis M, Zintzaras E. A network meta-analysis of randomized controlled trials of induction treatments in acute myeloid leukemia in the elderly[J]. Clin Ther, 2011, 33(3): 254-279.

[17]Ma YY, Zhou SJ, Chen FY, et al. Induction effects of decitabine in combination with valproic acid sodium on apoptosis of myeloma cells and its underlying mechanism[J]. Med J Chin PLA, 2013,38(10): 837-841. [馬泳泳, 周淑娟, 陳楓煜, 等. 俞康地西他濱聯(lián)合丙戊酸鈉誘導(dǎo)的骨髓瘤細(xì)胞凋亡及其機(jī)制研究[J]. 解放軍醫(yī)學(xué)雜志, 2013, 38(10): 837-841.]

[18]Lv HR, Deng Q, Li YM. Effect of Decitabine combined with atra on proliferation and differentiation of hl-60cells[J]. Tianjin Med J, 2013, 41(2): 154-157. [呂海容, 鄧琦, 李玉明. 地西他濱聯(lián)合全反式維甲酸對(duì)HL-60細(xì)胞增殖及分化的影響[J].天津醫(yī)藥, 2013, 41(2): 154-157.]

[19]Kantarjian HM, Thomas XG, Dmoszynska A, et al. Multicenter,randomized, open-label, phase III trial of decitabine versus patient choice, with physician advice, of either supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed acute myeloid leukemia[J]. J Clin Oncol, 2012,30(21): 2670-2677.

[20]Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study[J]. Cancer, 2006, 106(8): 1794-1803.

[21]Bordoni RE, Feinberg BA, Gilmore JW, et al. Hematologic outcomes of myelodysplastic syndromes treatment with hypomethylating agents in community practice[J]. Clin Lymphoma Myeloma Leuk, 2011, 11(4): 350-354.

[22]Cashen AF, Schiller GJ, O'Donnell MR, et al. Multicenter, phase II study of decitabine for the first-line treatment of older patients with acute myeloid leukemia[J]. J Clin Oncol, 2010, 28(4): 556-561.

[23]Blum W, Garzon R, Klisovic RB, et al. Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine[J]. Proc Natl Acad Sci USA, 2010, 107(16): 7473-7478.

[24]Wang LX, Mei ZY, Zhou JH, et al. Low dose decitabine treatment induces CD80expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses[J]. PLoS One, 2013,8(5): e62924.

[25]Turcan S, Fabius AW, Borodovsky A, et al. Efficient induction of differentiation and growth inhibition in IDH1mutant glioma cells by the DNMT Inhibitor Decitabine[J]. Oncotarget, 2013,4(10): 1729-1736.

[26]Tsai HC, Li H, Van Neste L, Cai Y, et al. Transient low doses of DNA-demethylating agents exert durable antitumor effects on hematological and epithelial tumor cells[J]. Cancer Cell, 2012,21(3): 430-446.

[27]Weng DS, Zhou J, Zhou QM, et al. Minimally invasive treatment combined with cytokine-induced killer cells therapy lower the short-term recurrence rates of hepatocellular carcinomas[J]. J Immunother, 2008, 31(1): 63-71.

[28]Shi M, Zhang B, Tang ZR,et al. Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma[J]. World J Gastroenterol,2004, 10(8): 1146-1151.

[29]M rten A, Ziske C, Sch ttker B, et al. Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both populations[J]. J Immunother, 2001, 24(6): 502-510.