CD56在急性髓系白血病细胞上的表达及微小残留病分析

引用本文

干灵红, 陈葆国, 颜荣文, 李伯利, 罗文达. CD56在急性髓系白血病细胞上的表达及微小残留病分析. 25(5): 360-364
GAN Linghong, CHEN Baoguo, YAN Rongwen, LI Boli, LUO Wenda. Expression of CD56 in the cells of acute myeloid leukemia and its clinical significance in minimal residual disease. Labratory Medicine, 25(5): 360-364 复制到剪切板

Permissions

《检验医学》编辑部

CD56在急性髓系白血病细胞上的表达及微小残留病分析

干灵红¹, 陈葆国¹, 颜荣文¹, 李伯利¹, 罗文达²

1. 浙江省台州医院中心实验室, 浙江临海 317000

2. 浙江省台州医院血液科, 浙江临海 317000

作者简介：干灵红,女,1968年生,学士,主治医师,主要从事血液肿瘤免疫学研究。

摘要

目的

探讨CD56在非M3急性髓系白血病(AML)中的表达及在微小残留病(MRD)检测的意义。

方法

应用流式细胞术分析145例非AML-M3 AML患者免疫表型,以CD56、CD19、CD117、CD34、CD33为标志,CD45设门MRD分析。

结果

145例AML患者中CD56阳性37例,占25.52%,CD19阳性17例,占11.72%,分布在M1、M2、M4、M5。CD56或/和CD19阳性的AML患者共49例,其中CD56和CD19同时阳性5例,均为M2亚型。P170蛋白阳性36例,占24.83%,其表达率与CD56呈显著正相关(P<0.01)。48例AML患者在诱导治疗结束和维持治疗第14、32、56周作MRD检测。单纯CD56⁺组MRD阳性率93.55%,复发率77.41%,与单纯CD19⁺组(MRD阳性率25.00%,复发率0)比较,差异有统计学意义(P均<0.01)。CD56⁺CD19⁺组MRD阳性率100.00%,复发率80.00%,与单纯CD56⁺组比较,差异无统计学意义(P>0.05),与单纯CD19⁺组比较,差异有统计学意义(P<0.01)。

结论

CD56、CD19在非M3 AML有较高的表达率。用CD45设门以CD56、CD19、CD34、CD117、CD33为组合的MRD方案能有效检测MRD。

关键词: CD56; 急性白血病; 微小残留病; 流式细胞术

中图分类号:R446.62 文献标志码:A 文章编号:1673-8640(2010)05-0360-05

Expression of CD56 in the cells of acute myeloid leukemia and its clinical significance in minimal residual disease

GAN Linghong¹, CHEN Baoguo¹, YAN Rongwen¹, LI Boli¹, LUO Wenda²

1.Central Laboratory, Taizhou Hospital of Zhejiang Province, Zhejiang Linhai 317000, China

2.Department of Hematology, Taizhou Hospital of Zhejiang Province, Zhejiang Linhai 317000,China

Abstract

Objective

To investigate the expression of CD56 in non-M3 acute myeloid leukemia (AML) and its clinical significance in the determination of minimal residual disease (MRD).

Methods

The immunophenotype of 145 patients with non AML-M3 AML was analyzed by flow cytometry as the markers of CD56, CD19, CD117, CD34 and CD33. MRD analysis was gated by CD45.

Results

CD56 and CD19 expressions were detected in 25.52% (37/145) and 11.72% (17/145) in patients with AML-M1, M2, M4 and M5, respectively. The data showed that 49 patients with AML were CD56 and /or CD19 positive , whose CD56 and CD19 both positive were all patients with AML-subtype M2 (5 cases). Meanwhile, P170 which was strongly associated with the CD56 expression was observed in 24.83% (36/145) of patients with AML (P<0.01). MRD was determined in 48 patients with AML after induction chemotherapy or at the 14^th, 32^th and 56^th week during the consolidation chemotherapy. MRD was observed in 93.55% of CD56 positive patients with AML, and the the relapse rate was 77.41%. The positive rate of MRD in CD19⁺ patients was 25.00% without relapse. There was statistical significance between the CD56⁺ and CD19⁺ patients with AML (P<0.01). The data also showed that MRD existed in all CD56⁺CD19⁺ patients with AML which the relapse rate was 80.00%. There was no significant difference with that of CD56⁺ patients with AML (P>0.05), and there was significant difference with that of CD19⁺ patients with AML (P<0.01).

Conclusions

CD56, CD19 express highly in non-M3 patients with AML. CD45 gating analysis with CD56, CD19, CD34, CD117, CD33 as a combination is an effective MRD diagnostic program.

Keyword: CD56; Acute myeloid leukemia; Minimal residual disease; Flow cytometry

Show Figures

白血病细胞的显著特征是抗原表达时相混乱, 出现抗原表达量异常、系列抗原交叉表达、抗原的跨阶段表达和光散射信号改变等, 称之为白血病相关免疫表型(leukemia associated immuno-phenotypes, LAIP)^[1]。流式细胞术利用 LAIP分析白血病微小残留病(MRD) 是MRD检测的主要方法之一。目前急性髓系白血病(AML)初治完全缓解(CR)率有显著提高, 但许多患者终因持续存在MRD而复发。有研究表明, CD56阳性的AML具有独特的临床生物学特征^[2], 主要表现为易髓外浸润、复发, 预后差, 而有效的MRD监测对其预后判断具有重要意义。有关AML中CD56 MRD检测方案国外有一些报道, 而国内尚无系统的研究报道。本研究检测非AML-M3 AML患者CD56 的表达, 探讨以CD56为中心联合其他免疫标志建立MRD检测方案, 并评估AML预后。

材料和方法

一、研究对象

2002年1月至2006年3月浙江省台州医院确诊并进行免疫分型检测的非AML-M3 AML患者145例, 其中男97例, 女48例, 中位年龄42(5~76)岁。细胞形态学(M)、免疫学(I)、细胞遗传学(C)即MIC分型: M0 8例, M1 27例, M2 34例, M4 29例, M5 38例, M6 9例。以10名健康骨髓供者和10名缺铁性贫血患者骨髓细胞作对照, 男14例, 女6例, 中位年龄37(25~46)岁。

二、材料

1.标本化疗前采骨髓2 5 mL肝素钠抗凝, 吸取100~500 μ L加2~5 mL不含固定剂的红细胞裂解液, 分离有核细胞, 用磷酸盐缓冲液制成细胞浓度为1× 10⁷/mL的单细胞悬液备用。

2.单克隆抗体 CD34、人类白细胞抗原DR(HLA-DR)、CD38、CD117、CD33、CD13、CD15、CD11b、CD64、CD14、髓过氧化物酶(MPO)、多药耐药蛋白(P170)、CD56、CD19、CD22、CD20、CD10、CD3、CD7、CD2、CD45等荧光[异硫氰酸荧光素(FITC)、藻红蛋白(PE)、叶绿素蛋白(PerCP)、别藻青蛋白(APC)]直标抗体均购自美国BD公司。

三、方法

1.抗体标记及流式细胞术免疫分型按多色分析法进行染色, 染前按100 μ L/10⁶细胞的比例加AB型血浆, 4 ℃培养20 min, 以阻断可结晶片段(Fc)受体。单克隆抗体染色方法按本室常规进行^[3]。应用FACSCalibur^TM流式细胞仪(美国BD公司)配套的Cell Quest 软件获取并分析10 000个细胞。通过CD45/侧散射光(SSC)设门识别幼稚细胞群。结果判断:CD34、P170> 10%, 其余抗原阳性细胞 > 20%为阳性。

2.MRD分析采用CD56/CD117/CD45/CD33、CD56/CD34/CD45/CD33、CD19/CD117/CD45/CD33、CD19/CD34/CD45/CD33 4种抗体组合方案进行MRD检测。Cell Quest 软件获取并分析10万30万个有核细胞, CD45/SSC设门, LAIP阳性细胞> 0.01%为MRD阳性, ≤ 0.01%为阴性, MRD> 0.01%为复发。

3.细胞遗传学检查治疗前取肝素抗凝骨髓5 mL, 采用24 h短期培养法制备染色体, G显带技术分析核型。根据《人类细胞遗传学国际命名体制(ISCN1995)》的有关规定描述核型。

四、统计学方法

应用SPSS 10.0软件包进行分析。计数资料采用χ ²检验(或Fisher确切概率法), 计量资料用非参数法两样本比较的秩和检验。直线相关分析指标的相关性。

结果

一、对照骨髓抗原表达

对照骨髓中有核细胞表达CD56⁺CD34⁺CD45⁺CD33⁺、CD56⁺CD117⁺CD45⁺ CD33⁺、CD19⁺CD117⁺CD45⁺ CD33⁺和CD19⁺CD34⁺CD45⁺ CD33⁺ 4种LAIP, LAIP阳性细胞中位数分别为0.005 6%、0.005 4%、0.005 5%和0.005 1%, 均< 0.01%。

二、初治AML患者抗原表达

1.145例AML患者中CD56阳性37例, 占25.52%, 分别分布在M1(5/27)、M2(18/34)、M4(4/29)、M5(10/38)。CD19阳性17例, 占11.72%, 分布在M1(1/27)、M2(13/34)、M4(1/29)、M5(2/38)。CD56或/和CD19阳性的AML患者共49例, 其中CD56和CD19同时阳性5例, 均是M2亚型。P170蛋白阳性36例, 占24.83%, 其表达率与CD56呈显著正相关(r=0.67, P< 0.01), 见表1。

表1 145例非AML-M3 AML 患者CD抗原表达情况

2.49例AML初诊患者骨髓细胞表达CD56⁺CD34⁺CD45⁺CD33⁺ 24例、CD56⁺CD117⁺CD45⁺CD33⁺ 25例、CD19⁺CD117⁺CD45⁺CD33⁺15例和CD19⁺CD34⁺CD45⁺ CD33⁺15例, 4种LAIP中LAIP阳性细胞中位数分别为38.14%、36.47%、32.08%和33.53%, 与对照组比较, 差异有统计学意义(P< 0.01)。见表2、图1。

表2 LAIP在对照骨髓和AML细胞上的表达比较[中位数(范围)]

	Figure Option View Download New Window
	图1 CD56⁺CD34⁺CD45⁺CD33⁺MRD分析注:ac为正常对照; df为AML初诊患者; gi为AML缓解患者

三、MRD及预后分析

49例CD56⁺或CD19⁺的AML患者中, 除1例失访外(单纯CD56⁺), 48例于诱导治疗结束和维持治疗第14、32、56周作MRD检测。将48例AML分成3组:单纯CD56⁺组, 单纯CD19⁺组, CD56⁺CD19⁺组。结果显示, 31例单纯CD56⁺组患者至56周时 MRD阳性29例(93.55%), 其中复发24例(77.41%), MRD阴性2例, 无复发。12例单纯CD19⁺患者至56周时 MRD阳性3例(25.00%), 9例阴性, 均无复发, 与单纯CD56⁺组比较, MRD阳性率差异有统计学意义(χ ²=17.90, P< 0.01), 复发率差异有统计学意义(χ ²=21.03, P< 0.01)。5例CD56⁺CD19⁺患者至56周时 MRD阳性5例(100.00%), 4例复发(80.00%), 与单纯CD56⁺组比较, MRD阳性率差异无统计学意义(χ ²=0.22, P=0.64), 复发率差异无统计学意义(χ ²=0.20, P=0.65); 与单纯CD19⁺组比较, MRD阳性率差异有统计学意义(Fisher确切概率法, P=0.009 0), 复发率差异有统计学意义(Fisher确切概率法, P=0.002 1)。

四、AML复发时LAIP转换

28例AML复发患者中免疫表型初诊阳性而复发表达缺失者为CD117(2/20)、CD34(4/23)、CD33(4/28)、CD56(3/28)、CD19(1/4)。

五、细胞遗传学分析

145例非AML-M3 AML患者中t(8; 21)阳性共17例, 分别分布在M2 14例、M4 2例、M5 1例。

讨论

CD56分子属超免疫球蛋白家族的成员, 是神经细胞黏附分子的异构体和自然杀伤淋巴细胞(NK)的标志。正常髓系原始细胞不表达CD56, 其在AML细胞出现属免疫表型系列交叉表达, 是LAIP中最具特异性的标志之一。有文献报道, CD56在AML中的表达频率为17%24%^[4]。CD56阳性的AML具有独特的临床生物学特征, 主要表现为易髓外浸润和复发, 生存期短, 因此检测CD56的表达及有效的MRD监测对临床治疗和预后判断具有重要意义。本研究结果显示, CD56在非AML-M3 AML的表达率25.52%, 以M1、M2、M4、M5为主; CD19也表达于AML, 占11.72%, 分布在M1、M2、M4、M5; 其中CD19和CD56同时阳性共5例, 均为M2亚型。

流式细胞术3~4色抗体组合检测MRD具有较好的敏感性和特异性, 尤其对急性淋巴细胞白血病可达10^-4, 而AML由于细胞异质性及骨髓中正常髓系幼稚细胞干扰, 只能达到10^-2~10^{-3 [1]}。近年来, Kern等^[1]研究发现, 在AML MRD抗体组合中加入CD45设门, 能够较好地排除正常幼稚细胞的干扰, 与非CD45设门组比较敏感性提高了一个对数级。本研究结果显示, 用CD45设门分析对照骨髓细胞敏感性达到10^-4。初诊AML患者的中位数分别为38.14%、36.47%、32.08%和33.53%, 表明该MRD方案具有较高的敏感性和特异性。

本组48例AML患者作了MRD检测。单纯CD56⁺组、CD56⁺CD19⁺组MRD阳性率、复发率明显高于单纯CD19⁺组。有3例CD56⁺患者出现2次复发, 同时高表达P170蛋白, 其中1例患者因化疗无法缓解而死亡。单纯CD19⁺组MRD阳性率只有25.00%, 3例MRD阳性患者随着巩固治疗逐渐转为阴性, 无1例复发。有文献报道CD56⁺AML易复发, 预后差, 生存期短, 既往认为CD19⁺AML往往伴随t(8; 21)染色体核型且预后良好^[5]。本研究结果支持上述观点。但是5例CD56⁺CD19⁺且t(8; 21)阳性的AML-M2患者却出现高频率的复发, 提示CD56⁺是主要原因。一方面可能由于CD56的神经黏附分子特性使得肿瘤细胞易于浸润; 另一方面, 我们发现CD56表达与多药耐药蛋白P170高表达呈正相关, 两者分子间可能存在某种联系, 有待进一步研究。

有文献报道, AML在复发时会发生免疫表型改变而影响MRD检测^[6]。Baer等^[2]发现CD56、CD19、CD14等在初诊患者低频率表达抗原会出现表达缺失, 而CD13、CD33、CD34等不曾表达的抗原可出现表达增加。本组复发的28例AML患者中CD117、CD34、CD33、CD56、CD19均有表达缺失。因此, 初诊时全面分析患者的免疫表型, 发现尽可能多的LAIP, 设计多组MRD抗体组合方案就显得尤为必要。本研究中3例CD56表达缺失的患者, 其中1例表达CD19, 通过CD19/CD34/CD45/CD33方案而获得MRD。

The authors have declared that no competing interests exist.

参考文献

View Option

[1]	Kern W, Voskova D, Schnittger S, et al. Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia[J]. Hematol J, 2004, 5(5): 410-418. [本文引用:3] [JCR: 1.857]
[2]	Baer MR, Stewart CC, Lawrence D, et al. Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21) (q22; q22)[J] . Blood, 1997, 90(4): 1643-1648. [本文引用:2] [JCR: 9.06]
[3]	陈葆国, 颜卫华, 孟哲锋, 等. CD66c在成人急性白血病细胞上的表达及意义[J]. 中华血液学杂志, 2006, 27(6): 370-373. [本文引用:2]
[4]	Auewarakul CU, Promsuwicha O, U-Pratya Y, et al. Immunophenotypic profile of adult acute myeloid leukemia (AML): analysis of 267 cases in Thailand [J]. Asian Pac J Allergy Immunol, 2003, 21(3): 153-160. [本文引用:1]
[5]	Ferrara F, Di Noto R, Annunziata M, et al. Immuno-phenotypic analysis enables the correct prediction of t (8;21) in acute myeloid leukaemia[J]. Br J Haematol, 1998, 102(2): 444-448. [本文引用:1]
[6]	Voskova D, Schoch C, Schnittger S, et al. Stability of leukemia-associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: comparison with cytomorphologic, cytogenetic, and molecular genetic findings[J]. Cytometry B Clin Cytom, 2004, 62(1): 25-38. [本文引用:1] [JCR: 2.231]

2004

1.857

0.0

. 2004, 5(5):null-null

Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia

Nature - the world's best science and medicine on your desktop

... 白血病细胞的显著特征是抗原表达时相混乱,出现抗原表达量异常、系列抗原交叉表达、抗原的跨阶段表达和光散射信号改变等, 称之为白血病相关免疫表型(leukemia associated immuno-phenotypes, LAIP)^[1] ...

... 流式细胞术3~4色抗体组合检测MRD具有较好的敏感性和特异性,尤其对急性淋巴细胞白血病可达10^-4,而AML由于细胞异质性及骨髓中正常髓系幼稚细胞干扰,只能达到10^-2~10^{-3 [1]} ...

... 近年来,Kern等^[1]研究发现,在AML MRD抗体组合中加入CD45设门,能够较好地排除正常幼稚细胞的干扰,与非CD45设门组比较敏感性提高了一个对数级 ...

1997

9.06

0.0

... 有研究表明,CD56阳性的AML具有独特的临床生物学特征^[2],主要表现为易髓外浸润、复发,预后差,而有效的MRD监测对其预后判断具有重要意义 ...

... Baer等^[2]发现CD56、CD19、CD14等在初诊患者低频率表达抗原会出现表达缺失,而CD13、CD33、CD34等不曾表达的抗原可出现表达增加 ...

2006

0.0

... 单克隆抗体染色方法按本室常规进行^[3] ...

2003

0.0

... 有文献报道,CD56在AML中的表达频率为17%24%^[4] ...

1998

0.0

. 1998, 102(2):444-448

Immunophenotypic analysis enables the correct prediction of t(8;21) in acute myeloid leukaemia

Felicetto Ferrara 1 ,Rosa Di Noto 1 ,Mario Annunziata 1 ,Carolina Copia 1 ,Catia LO Pardo 2 ,Piernicola Boccuni 2 ,Lucia Sebastio 3 andLuigi Del Vecchio 2

1 Divisione di Ematologia, 2 Servizio di Immunoematologia, 3 Servizio di Genetica Medica, Ospedale A. Cardarelli, Napoli, Italy * Dr FelicettoFerrara Via Pigna 76 F/G, 80128 Napoli, Italy.

> Immunophenotypic findings from 14 patients affected by acute myeloid leukaemia (AML) with t(8;21) were compared to those obtained from 79 AML patients with normal or other aberrant karyotypes. Classic lineage markers, adhesion molecules, surface enzymes, stem-cell- related antigens and HLA-DR were investigated. Following evaluation by the Mann-Whitney test, we found that t(8;21) AMLs showed a significantly higher expression of CD19, CD34, CD56, CD45RA and CD54. Conversely, blasts from patients in the control group significantly expressed higher levels of CD45R0, CD33, CD36, CD11b and CD14. In order to split the data at the best cut-off point to achieve the most homogenous subset with regard to cytogenetic pattern, i.e. t(8;21) or not, the CART (Classification and Regression Trees) method was applied. In the univariate analysis by CART, statistically significant differences were found when CD19 was dichotomized at 10%, CD34 at 37%, CD45RA at 84%, CD54 at 21%, CD56 at 12% , CD36 at 14%, CD45R0 at 25%, CD11b at 18% and CD14 at 12%. Once cut-off points were established by CART, we applied the logistic regression model to establish which combination of two or more antigens was most predictive for t(8;21). The combination CD19-CD34 at the cut-off points indicated above correctly classified 92/93 cases (98.9%). The addition of any other antigen combination to the CD19/CD34 model failed to improve the level of prediction. We conclude that AML with t(8;21) displays an exclusive immunophenotype that is highly predictive of the cytogenetic pattern.

... 21)染色体核型且预后良好^[5] ...

2004

2.231

0.0

... 有文献报道,AML在复发时会发生免疫表型改变而影响MRD检测^[6] ...