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)劑量的地西他濱對老年患者的骨髓毒性作用使其應(yīng)用受到很大限制。對于老年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]。考慮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)。患者從2008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波動于77～138g/L。血小板在第2個療程開始上升，第6個療程達(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%。療效評價達(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個與總生存相關(guān)的預(yù)后指標(biāo)，其中，大于3個指標(biāo)的患者中位總生存期只有1個月。根據(jù)此預(yù)后評價標(biāo)準(zhǔn)，本例預(yù)期生存期為1個月(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個月，治療相關(guān)死亡率13%～25%[15,22-23](表3)。但對于由MDS轉(zhuǎn)化而來的80歲以上高齡AML患者，當(dāng)前報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個周期，患者生存期達(dá)22個月，顯著高于文獻(xiàn)報道。對于本方案的療效機(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主要顯示細(xì)胞毒作用，而在低劑量時則表現(xiàn)為對腫瘤相關(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)胞對腫瘤細(xì)胞的識別和殺傷；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。

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。

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