应用荧光原位杂交技术检测84例慢性淋巴细胞白血病遗传学异常及预后分析

 摘要 目的：探讨荧光原位杂交 (fluorescence in situ hybridization,FISH) 技术检测慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者的遗传学异常,并分析与其他预后指标的相关性及预后价值。方法：回顾性分析采用FISH技术进行着丝粒探针CSP12(12p11.1～12q11.1)和序列特异性探针D13s25(13q14.3)、ATM (11q22.3)、RB1(13q14)、p53(17p13.1)检测过的84例CLL患者的临床以及实验室资料。分析FISH技术检测CLL患者中遗传学异常的发生率,并分析遗传学异常与CLL患者年龄、性别、Binet分期、血清乳酸脱氢酶(lactate dehydrogenase,LDH)、CD38表达、生存期之间的相关性。结果：(1)84例CLL患者中,62例有至少有一种遗传学异常。遗传学异常的检出率为73.8%。(2)共发现5种遗传学异常,依次为13q14缺失（56%）、P53缺失（28.6%）、-12（16.7%）、ATM缺失（13.1%）、+12（13.1%）。其中-12为CLL病人中新发现遗传学异常。(3)分析上述5种遗传学异常与年龄、性别、Binet分期、LDH水平、CD38表达的关系,在>60岁年龄病人里,+12阳性率显著高于<60岁病人(24%VS3%, P=0.042),与性别、Bient分期、LDH水平、CD38表达无关。(4)在LDH增高组中,CD38高表达的病例比例增高（67%VS20%, P=0.015）。(5) 伴有del(17p13)或del(11q22)或复杂异常中位生存时间为75个月,单纯具有del(13q14)异常组的中位生存时间为150个月,其他中位生存时间为120月,但两两比较没有达到统计学差异。结论：(1)FISH技术是一种在分析CLL患者遗传学异常方面较为快速、准确和敏感的方法; (2)CLL病人中除了常见13q14缺失、P53缺失、ATM缺失、+12之外,还可以出现-12异常。     

经典型Richter综合征转化前后IgVH基因克隆重排及突变分析

目的 检测经典型Richter综合征的IgVH基因克隆重排及突变状态,分析转化前后IgVH基因的分子特征及其与预后的关系,并对可能涉及的分子机制进行初步探讨.方法 用基因扫描和测序分析IgVH基因.另外用免疫组织化学LAB-SA法检测两种肿瘤中zeta链结合蛋白激酶70(ZAP70)、p53、干扰紊调节因子(IRF)-4等可能潜在危险因子的表达,并结合随访资料进行生存率分析,筛选危险因子.结果 (1)B-CLL/DLBCL克隆相关(18/23,78.3%),克隆不相关(5/23,21.7%);(2)在16例克隆相关中,12例转化前B-CLL及转化后DLBCL携带未突变IgVH基因;(3)转化前后IgVH基因使用是非随机的,在克隆相关的病例中,B-CLL/DLBCL最常使用VH3-23、VH3-74、VH1-2,各占11.1%;(4)转化后DLBCL仅部分表达CD5(32.1%)和CD23(14.3%)及ZAP70(23.8%),绝大多数表达p53(80.6%)和IRF-4(82.6%);(5)17例转化后DLBCL患者中位生存时间为7个月;(6)统计学分析生存时间与B-CLl/DLBCL转化前后克隆相关与否、IgVH基因的突变状态、ZAP70、p53、IRF-4的表达不相关.结论 (1)转化后DLBCL中克隆相关与克隆不相关的比例为2:1;(2)克隆相关的DLBCL多由携带未突变型IgVH基因的B-CLL患者转化而来;(3)发生转化的B-CLL中IgVH基因使用的偏向性提示了抗原在转化中的可能作用;(4)转化后DLBCL与普通DLBCL在IgVH基因的使用、突变状态,免疫表型及预后的不同,提示其可能是一种新的DLBCL亚类.     

一例伴有der(Y)t(Y;1)异常的多发性骨髓瘤的临床和实验研究

目的 对1例伴有不平衡染色体易位der(Y)t(Y;1)的多发性骨髓瘤(multiple myeloma,MM)患者进行细胞遗传学、中期荧光原位杂交、免疫学及临床研究.方法 采用细胞遗传学G显带行中期染色体核型分析;用1号染色体涂抹探针、Y染色体异染色质区探针进行中期荧光原位杂交检测;免疫分型检测CD38、CD138、ZAP70等的表达及免疫电泳检测免疫球蛋白类型等.结果 细胞遗传学分析结果 发现患者具有高度复杂的异常克隆,其核型为:92,XXYY[3]/49,X,der(Y)t(Y;1)(q12;q21),t(11;14)(q13;q32),+18,+20,+21[47]/49,X,idem,del(13q22),ace[1]/98,XX,der(Y)t(Y;1)×2,+18,+18,+20,+20,+21,+21[10]/46,XY[19].中期荧光原位杂交结果 证实der(Y)t(Y;1)的G显带结果 ,为1q部分三体与Y染色体长臂的不平衡易位.其异常的单克隆免疫球蛋白为IgD,定量6.24 g/L;免疫分型结果 为表达CD38、CD138,不表达ZAP70,考虑为异常克隆浆细胞的表达.结论 Y染色体的结构异常在血液系统肿瘤中非常罕见,本文报道1例发生于多发性骨髓瘤中的伴der(Y)t(Y;1)的核型异常、实验室及临床特点.     

荧光原位杂交技术检测多发性骨髓瘤分子遗传学改变及其意义

目的 研究多发性骨髓瘤(multiple myeloma,MM)患者1q21扩增、13q14缺失、TP53基因缺失及IgH易位的发生率及预后意义.方法 应用CD138单克隆抗体磁珠分选结合间期荧光原位杂交技术检测43例MM患者上述各种核型异常.结果 43例患者中28例(65.1%)出现了1q21的扩增,30例(69.7%)出现了13q14的缺失,8例(18.6%)出现了TP53基因的缺失,29例(67.4%)出现了IgH易位.1q21扩增、13q14缺失和TP53基因缺失与MM高死亡率有密切相关性.结论 1q21扩增、13q14缺失、TP53基因缺失及IgH易位是MM常见的核型异常,1q21扩增、13q14缺失和TP53基因缺失是MM的预后不良因子.     

河南地区不育症患者Y染色体微缺失和染色体异常的临床研究

目的探讨河南地区Y染色体AZF微缺失和染色体核型异常在无精子症、弱少精子症不育患者中的临床意义。方法采用PCR技术、染色体核型分析技术对2015年8月至2017年7月来本院诊治的906例男性不育患者,进行外周血染色体分析及Y染色体(AZF基因)微缺失检测。结果 906例样本Y染色体AZF微缺失检测和染色体异常异常率分别为10.5%(96/906)和6.3%(57/906)。AZF区缺失高频发生于AZFc+d(152)区,占总缺失的70.8%(68/96)。染色体核型分析检出性染色体异常31例、染色体结构(平衡易位)异常2例、染色体多态24例。结论染色体核型异常与Y染色体微缺失是男性无精子症、少精子症患者的重要病因,对于生殖异常的男性行外周血染色体检查和AZF微缺失检测有助于明确其遗传学病因,为男性不育临床诊断和治疗提供科学依据。     

DSP30联合IL-2刺激在伴有骨髓侵犯的B细胞非霍奇金淋巴瘤常规染色体核型分析中的应用

目的研究非甲基化胞嘧啶鸟嘌呤二核苷酸寡脱氧核苷酸(CpG-ODN, DSP30)在伴有骨髓浸润的B细胞非霍奇金淋巴瘤(B cell lymphoma, B-NHL)常规染色体检测中的价值。方法搜集116例初诊时伴有骨髓浸润的B-NHL标本, 设实验组应用DSP30联合IL-2刺激细胞增殖并培养72 h后进行染色体制备;对照组常规24 h培养处理并制备染色体, 运用R显带技术对患者骨髓标本进行核型分析。结果 116例B-NHL中慢性淋巴细胞白血病/小淋巴细胞淋巴瘤(CLL/SLL)47例, 弥漫大B细胞淋巴瘤(DLBCL)19例, 套细胞淋巴瘤(MCL)16例, 滤泡细胞淋巴瘤(FL)13例, 边缘区淋巴瘤(MZL)8例, 伯基特淋巴瘤(BL)7例, 淋巴浆细胞淋巴瘤/华氏巨球蛋白血症(LPL/WM)3例, 毛细胞白血病(HCL)2例, 富于T/组织细胞的大B细胞淋巴瘤1例;对照组116例行常规染色体检测, 18例无分裂相, 染色体成功分析中异常检出率为22.45%(22/98), 而经实验组(DSP30+IL-2)刺激的同期患者, 无分裂相者减少至10例, 异常检出率为59.43%(6...     

荧光原位杂交技术在产前诊断的应用价值

目的研究荧光原位杂交(FISH)技术对诊断胎儿染色体异常的临床应用。方法有654例孕妇自2016年6月至2017年7月在我院进行产前诊断,利用FISH技术和细胞染色体核型分析对获取的脐带血细胞遗传物质的分析。结果 654例孕妇经细胞染色体核型分析和FISH技术均发现16例为21-三体综合症;8例为18-三体综合症;染色体核型分析发现平衡易位6例,倒位2例,FISH技术未检出染色体结构异常。结论 FISH技术对胎儿染色体数目异常敏感性较高,对于染色体结构变异的检测有一定局限性。     

羟氯喹对CpG-ODN诱导浆细胞样树突状细胞活化影响的初步研究

目的体外探讨羟氯喹(HCQ)对CpG-ODN诱导pDCs活化的影响。方法产房收集脐带血250~300 ml,密度梯度离心得脐带血单核细胞,BDCA-4磁珠分选得前体pDCs细胞,前体pDCs分为3组(IL-3,IL-3+CpG,IL-3+CpG+HCQ)培养3 d,流式细胞术测pDCs表面BDCA-2、CD86、CD32表达,ELISA检测上清细胞因子IFN-α水平。结果①IL-3+CpG组BDCA-2表达水平和荧光强度均显著低于IL-3组(P<0.05);CD86和CD32表达水平、荧光强度和培养上清IFN-α浓度显著高于IL-3组(P<0.05)。②IL-3+CpG+HCQ组CD32表达水平和荧光强度均显著低于IL-3+CpG组(P<0.05);培养上清IFN-α浓度显著低于IL-3+CpG组(P<0.05)。结论 CpG-ODN可以活化pDCs,活化的pDCs高表达CD86、CD32,低表达BDCA-2;HCQ抑制CD32表达,抑制CpG-ODN诱导的pDCs活化,减少IFN-α的分泌。     

产前诊断中FISH鉴定染色体平衡易位来源研究

目的探讨利用FISH鉴定染色体平衡易位及其来源在产前诊断中的应用。方法常规羊水细胞培养和染色体制备分析,最后采用荧光原位杂交技术进行鉴定。结果常规G显带(320条带)下分析胎儿的染色体核型为46,XX,?17q25mat;最后FISH鉴定为ish der(11)t(11;17)(p14.3;q25)(11pter-,17qter+,11qter+)mat,der(17)t(11;17)(p14.3;q25)(17pter+,17qter-,11pter+)mat。结论 FISH能够弥补常规G显带条件下对亚显微结构异常难以鉴定的不足,以明确胎儿染色体结构异常的来源。     

男性不育症与染色体异常和Y染色体微缺失的临床分析

目的探讨男性不育症与染色体异常和Y染色体微缺失的相关性。方法 2017年1月-2018年12月在我院就诊的412例诊断为不育症男性患者进行外周血染色体核型和Y染色体微缺失检查。结果 412例男性不育症患者共检出异常染色体核型34例,异常率8.25%,主要表现为染色体多态性和平衡易位,其中:①染色体多态性32例,占94.12%;②染色体平衡易位2例,占5.88%。412例男性不育症患者共检出Y染色体微缺失26例,发生率6.31%,其中AZFa区缺失4例,AZFb区缺失6例,AZFc区缺失12例,AZFd区缺失1例,AZFa+AZFc区缺失1例,AZFb+AZFc区缺失1例,AZFa+AZFb+AZFc区缺失1例。结论染色体异常和Y染色体微缺失在男性不育症患者发生率较高,作为常规检查有助于明确其遗传学病因,为遗传咨询提供科学依据。     

Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: a CLL Research Consortium (CRC) Study

Cytogenetic abnormalities are important prognostic indicators in CLL. Historically, only interphase cytogenetics was clinically useful in CLL, because traditional mitogens are not effective mitotic stimulants. Recently, CpG-oligodeoxynucleotide (ODN) stimulation has shown effectiveness in CLL cells. The CLL Research Consortium tested the effectiveness and reproducibility of CpG-ODN stimulation for detecting chromosomally abnormal clones by five laboratories. More clonal abnormalities were observed after culture of CLL cells with CpG-ODN than with the traditional pokeweed mitogen plus 12-O-tetradecanoylphorbol-13-acetate (PWM+TPA). All clonal abnormalities in PWM+TPA cultures were observed in CpG-ODN cultures, whereas CpG-ODN identified some clones not found by PWM+TPA. CpG-ODN stimulation of one normal control sample and 12 CLL samples showed that, excepting clones of del(13q) in low frequencies and one translocation, results in all five laboratories were consistent, and all abnormalities were concordant with FISH. Abnormal clones in CLL were more readily detected with CpG-ODN stimulation than with traditional B-cell mitogens. With CpG-ODN stimulation, abnormalities were reproducible among cytogenetic laboratories. CpG-ODN did not appear to induce aberrations in cell culture, but did enhance detection of abnormalities and complexity in CLL. Because karyotypic complexity is prognostic and is not detectable by standard FISH analyses, stimulation with CpG-ODN is useful for identifying this additional prognostic factor in CLL.     

Zap-70 and CD38 as predictors of IgVH mutation in CLL

Zeta-chain (TCR)-associated protein kinase 70 kDa (Zap-70) and CD38 expression may be of prognostic significance in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap-70 and CD38 are usually positive in cases of CLL with unmutated immunoglobulin variable region genes (IgVH) and may be used to predict IgVH mutation status and prognosis. Usually cases of CLL positive for Zap-70 or CD38 indicate a worse prognosis. In the present investigation, 47 cases of CLL were evaluated for CD38 expression, and 17 cases were evaluated for both Zap-70 and CD38 expression. Of the 47 cases, 19 (40.4%) positively expressed CD38. Of the 17 cases evaluated for Zap-70, 11 (64.7%) were positive for Zap-70, while only 6 (35.3%) were positive for CD38 expression; the remaining cases were negative for CD38. The results of this study show that Zap-70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a more extensive study to evaluate the significance of Zap-70 and CD38 expression as indicators of IgVH mutation status and prognosis of CLL patients.     

ZAP70 expression assessed by immunohistochemistry on peripheral blood: a simple prognostic assay for patients with chronic lymphocytic leukemia.

The somatic hypermutational (SHM) status of the immunoglobulin heavy-chain variable (IgVH) gene is a powerful prognostic factor in patients with chronic lymphocytic leukemia (CLL). However, IgVH SHM analysis is not well-suited to routine use in the clinical diagnostic laboratory. ZAP70 expression is a potential surrogate for the absence of SHM. Given the current problems with the standardization of ZAP70 assessment by flow cytometry, we sought an alternative approach, using immunohistochemistry (IHC). The utility of IHC is largely restricted to tissues, precluding its routine application to most patients with CLL who are typically diagnosed based upon peripheral blood (PB) findings. Accordingly, we developed an IHC assay that can be performed on PB. Enriched PB mononuclear cells from 29 patients with CLL were analyzed for ZAP70 expression by IHC on paraffin-embedded cell blocks, using standard techniques. IgVH SHM analysis was performed on all cases, and clinical features recorded. Seventeen specimens (59%) were negative for ZAP70 expression and 12 (41%) were positive for ZAP70 expression. SHM was evident in 20 specimens (69%), and absent in 9 (31%). Seventy-six percent of the specimens (22/29) displayed "concordant" ZAP70 and SHM results, in that 15 (52%) were SHM-positive/ZAP70 negative, whereas 7 (24%) were SHM-negative/ZAP70 positive. ZAP70 expression in this small cohort correlated with poor clinical outcome. Importantly, IHC analysis of ZAP70 in PB is a simple, reliable, robust assay that may have a valuable role in the routine clinical laboratory assessment of patients with CLL.     

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.     

Intracellular cytokine expression in T cells from patients with chronic lymphocytic leukemia

Functional disorders of T lymphocytes play an essential role in abnormal immune response in patients with chronic lymphocytic leukemia. The aim of this study was to assess the profile of cytokines expressed by T cells derived from patients with CLL. We have demonstrated that the intracellular levels of IL-2, IL-4, IFN-γ, TNF, IL-6 and IL-10 were significantly higher in T cells of CLL patients than in healthy donors. Moreover, the percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+, and CD4+/CD3+/IL-2+ cells were significantly higher in ZAP-70-positive patients compared with ZAP-70-negative ones. Likewise, significantly higher percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-γ+ cells were observed in CD38-positive than in CD38-negative cases. What is more, there was a significant difference in median percentage of CD3+/CD4+ cells expressing TNF, IL-4, IFN-γ, IL-2 or IL-6 between patients carrying the 11q22.3 deletion and/or the 17p13.1 deletion and patients without these genetic aberrations. Our results confirm the functional disorders of T cells in CLL and their influence on the clinical course of the disease.     

Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.     

应用组合荧光原位杂交技术检测慢性淋巴细胞白血病的基因组异常

目的 探讨应用组合荧光原位杂交(panel fluorescence in situ hybridization，panel FISH)技术对慢性淋巴细胞性白血病(chronic lymphocytic leukaemia，CLL)基因组异常检测的价值。方法 分别应用3号、12号、18号的着丝粒探针和序列探针D13S272(13q14．3)、ATM(11q23)等5种荧光素标记的DNA探针，对22例CLL患者进行FISH检测，并和常规细胞遗传学检测结果进行比较，以确定何种方法对检测CLL的染色体和基因组异常更为敏感可靠。结果 22例CLL患者中，常规细胞遗传学检测出8例(36．3％)有染色体异常，包括单纯 123例； 3、 12l例； 3、 12、 18 1例；t(5；15)1例；13q-1例；3q-、18p 1例；4q 和13q-1例；组合FISH检测出15例(68，1％)有染色体异常，包括 34例、 126例、 18l例、11q-6例、13q-8例。结论组合。FISH技术是检测CLL患者染色体基因组异常的有效手段，与常规细胞遗传学方法相结合则可明显提高CLL染色体异常的检出率。     

Burkitt t(8;14)(q24;q32) and cryptic deletion in a CLL patient: report of a case and review of literature

A 53-year-old man diagnosed with chronic lymphocytic leukemia (CLL)-small lymphoma following splenectomy was found to have a t(8;14) with an apparent cryptic deletion of the MYC gene. This patient's spleen and bone marrow (BM) showed that 93% and approximately 70% of the viable cells, respectively, were lambda-monoclonal B-cells coexpressing CD5 with CD20, CD19, CD23, CD22, CD38, and low FMC-7. The smear showed a marked increase in small, mature lymphoid cells, with <2% prolymphocytes. The BM karyotype was 46,XY,t(8;14)(q24;q32),-18,+mar[3]/46,XY[27] and FISH analysis with an IGH/MYC green-red dual-fusion signal probe showed an atypical interphase result of one fusion, two green, and one red signal in 70% of the cells. The MYC dual red-green split-apart probe showed the expected t(8;14) pattern in 62% of the cells; however, sequential FISH on a t(8;14) GTG-metaphase showed the single fusion signal on derivative chromosome 8 and only a green signal on der(14) for the IGH/MYC dual-fusion probe and a green signal on der(14), red signal on der(8), and fusion signal on the normal chromosome 8 for the MYC split-apart probe. Thus, the apparently balanced t(8;14) had a cryptic deletion (approximately 1.6 Mb) between the red and the green regions flanking the MYC gene in the MYC split-apart probe, 128,585,631-130,226,339 bp from 8pter. The rarity of t(8;14) in CLL together with a cryptic deletion that probably includes the MYC gene in our CLL patient persuaded us to explore the clinicopathological role of MYC translocations by comparing disease progression in our patient and in other reported CLL cases.