V(D)J-mediated translocations in lymphoid neoplasms: a functional assessment of genomic instability by cryptic sites.

Most lymphoid malignancies are initiated by specific chromosomal translocations between immunoglobulin (Ig)/T cell receptor (TCR) gene segments and cellular proto-oncogenes. In many cases, illegitimate V(D)J recombination has been proposed to be involved in the translocation process, but this has never been functionally established. Using extra-chromosomal recombination assays, we determined the ability of several proto-oncogenes to target V(D)J recombination, and assessed the impact of their recombinogenic potential on translocation rates in vivo. Our data support the involvement of 2 distinct mechanisms: translocations involving LMO2, TAL2, and TAL1 in T cell acute lymphoblastic leukemia (T-ALL), are compatible with illegitimate V(D)J recombination between a TCR locus and a proto-oncogene locus bearing a fortuitous but functional recombination site (type 1); in contrast, translocations involving BCL1 and BCL2 in B cell non-Hodgkin's lymphomas (B-NHL), are compatible with a process in which only the IgH locus breaks are mediated by V(D)J recombination (type 2). Most importantly, we show that the t(11;14)(p13;q32) translocation involving LMO2 is present at strikingly high frequency in normal human thymus, and that the recombinogenic potential conferred by the LMO2 cryptic site is directly predictive of the in vivo level of translocation at that locus. These findings provide new insights into the regulation forces acting upon genomic instability in B and T cell tumorigenesis.     

急性混合细胞白血病伴t(12;22)一例报告--附文献复习

目的报道1例伴有t（12;22）（p13;q12）的急性混合细胞白血病.方法骨髓细胞经24h短期培养后按常规方法制备染色体,采用R显带技术进行细胞遗传学分析.应用抗生物素蛋白生物素复合物（ABC）法和单克隆抗体检测白血病细胞的表面抗原;12号和22号全染色体涂染探针分别以绿色和红色2种荧光素标记后进行双色荧光原位杂交（FISH）涂染检测.结果患者的临床表现和实验室检查均符合急性混合细胞白血病.免疫表型分析髓系和淋系标记均呈阳性;染色体核型为46,XX,t（12;22）（p13;q12）[6]/46,XX,idem,der（2）[2]/46,XX[12].骨髓中期细胞经双色FISH证实12号染色体短臂和22号染色体长臂之间发生了易位.结论t（12;22）（p13;q11-13）是恶性血液病中少见的染色体异常.t（12;22）患者具有独特的临床、细胞遗传学和分子生物学特点.该染色体异常对急性白血病的预后判断价值仍需进一步观察.     

两例急性髓系白血病伴t(6;21;8)(p22;q22;q22)复杂易位患者的临床与实验室研究

目的探讨2例急性髓系白血病（AML）伴t（6;21;8）（p22;q22;q22）复杂易位患者的临床及实验室特点.方法骨髓细胞经短期24 h培养后按常规方法制备染色体标本,R显带进行核型分析;双色双融合AML1/ETO探针进行丝裂间期及中期荧光原位杂交（FISH）检测AML1/ETO融合信号;逆转录-聚合酶链反应（RT-PCR）检测AML1/ETO融合基因转录本;综合分析临床特征.结果2例患者常规细胞遗传学分析显示均存在t（6;21;8）（p22;q22;q22）,间期和中期FISH证实了核型结果;RT-PCR检测到AML1/ETO融合基因转录本;尽管2例患者均诊断为AML-M2,但二者的免疫表型和治疗反应不同.结论t（6;21;8）（p22;q22;q22）是一种少见的t（8;21）（q22;q22）的复杂变异易位,还需要更多的病例以明确其临床特征和预后价值.     

Notch与恶性血液病研究进展

Notch信号通路由Notch受体、配体和胞内的效应分子组成.1991年,第1个人类Notch家族成员Notch1 基因在具有t(7;9) (q34;q34.3)染色体易位的急性T淋巴细胞白血病中发现,由此揭示了Notch信号通路是一条影响细胞命运的保守而重要的信号通路,几乎涉及所有细胞的增殖、分化和凋亡.与此同时,Notch信号通路在恶性血液病和实体瘤的形成和发展中所起的作用也日趋清晰.在超过半数的急性T淋巴细胞白血病中发现了突变型Notch1基因,但其作为原发还是继发仍不确定.近年来的研究发现,Notch信号通路具有致癌和抑癌双重作用.现就Notch信号通路在不同类型恶性血液病发生和发展中所起的作用、急性T淋巴细胞白血病中突变型Notch1基因类型、Notch信号通路与其它相关信号通路的关系及Notch信号通路抑制剂在恶性血液病治疗中的作用等相关研究进展进行综述.     

9例γδT细胞淋巴瘤/白血病患者的临床及实验室特征

目的 报告9例γδ T细胞淋巴瘤/白血病患者的临床及实验室特征.方法 对2007至2010年住院诊治的患者,常规询问病史,进行体检及实验室检测.对有异常细胞的骨髓或脾组织用流式细胞分析技术(FCM)行免疫分型,同时用PCR法检测TCRγ、TCRδ基因克隆性重排,用G显带技术分析染色体,用多重筑巢式PCR技术筛查急性白血病基因及1-8型人类疱疹病毒基因,并行形态学及细胞化学染色.根据表达TCRγδ链来确定γδT细胞,根据T细胞抗原表达异常确定恶性γδT细胞,根据γδT细胞表达CD34、CD99、TDT、CD1a及急性白血病基因等确定为前体γδT细胞.结果 共9例患者经FCM分析诊断为γδT细胞淋巴瘤/白血病,初诊时8例患者骨髓中检测出大量恶性细胞,细胞形态同原始细胞.5例患者诊断为急性T淋巴细胞白血病(T-ALL)或淋巴母细胞淋巴瘤(LBL)(γδT型),4例患者诊断为肝脾γδT细胞淋巴瘤(HSγδTCL).行TCR基因克隆重排检测的6例患者均检测出有克隆性TCRγ和(或)TCRδ基因重排.全部患者化疗难以完全缓解(CR)或CR后很快复发,5例接受异基因造血干细胞移植(allo-HSCT)的患者4例持续CR,CR时间分别为2、2、3、12个月;1例TALL(γδT型)患者在allo-HSCT后1个月内复发.结论 γδT细胞淋巴瘤/白血病的发病率可能比既往报道的高,部分T-ALL/LBL也为γδT细胞型;FCM是快速可靠的诊断方法,克隆性TCRγ和(或)TCRδ基因重排阳性有助于诊断;患者预后差,allo-HSCT可能是唯一可治愈的方法.

Abstract：

Objective To analyze the clinical and laboratory features of 9 cases of γδT cell lymphoma or leukemia.Methods From 2007 to 2011, 9 patients with γδT-cell lymphoma/leukemia were diagnosed in our hospital.The immunophenotype of the abnormal cells were detected by flow cytometry, clonal gene rearrangement of IgH,TCRγ,TCRδ by PCR, chromosome karyotype analysis by G banding, acute leukemia gene and the DNA of type 1-8 human herpes virus by mulplex nested PCR, The γδT cells were determined by T cell with TCR γδ chain, the malignant γδ T cells by the abnormal expression of T cell antigens and the precursor malignant γδ T cells by the expression of CD34, TDT ,CD99, CD1a or acute leukemia genes.Results In the 9 patients with γδT cell lymphoma leukemia, significant malignantγδT cells infiltration of bone marrow were found in 8 with blast morphology.5 were diagnosed as T-ALL/LBL(γδT type) and 4 HSγδ TCL.The clonal gene rearrangement of TCRγ and/or TCRδ were detected in 6/6 patients.Patients either did not achieve complete remission (CR) after induction therapy or relapsed quickly after CR.Only 4/5 patients remained continuous CR(CCR) at 2,2,3,12 months respectively, after allogeneic hematopoietic stem cell transplantation(allo-HSCT), the fifth T-ALL(γδT) relapsed 1 month after allo-HSCT .Conclusion s The incidence of γδ T cell lymphoma or leukemia may be higher than reported, part of them were T-ALL/LBL with poor prognoses.FCM and clonal gene rearrangement of TCRγ and/or TCRδ are helpful to diagnosis.Allo-HSCT may be the only curative approach.     

二例伴有t(3;5)(q25;q34)的骨髓增生异常综合征的临床和实验研究

目的：报道2例伴有t(3；5）（q25；q34）的骨髓增生异常综合征（MDS）。方法：骨髓细胞24h培养后按常规方法制备染色体，用R显带技术进行细胞遗传学分析，并以3号和5号染色体涂染探针进行荧光原位杂交（FISH）检测。结果：2例的临床和血液学改变符合MDS诊断，染色体核型分析揭示2例患者均有一致的核型异常：46，XY，t(3；5）（q25；q34）；其中1例患者的双色FISH检测证实1条3号染色体长臂和1条5号染色体长臂之间发生了相互易位。结论：t(3；5）（q25；q34）是一种少见的核型异常，它和MD有特别的联系，常有涉及三系的病态造血改变；染色体涂染技术是检测该易位的可靠手段。     

The chromosome translocation (11;14)(p13;q11) associated with T cell acute leukemia. Asymmetric diversification of the translocational junctions

The t(11;14)(p13;q13) translocation associated with T cell acute lymphocytic leukemia generates two abnormal chromosomes, designated 11p+ and 14q-. To investigate the mechanism of t(11;14)(p13;q11) formation, we analyzed the translocation junctions of 11p+ and 14q- from two patients. The 11p+ junctions consisted of precise fusions of a pseudo recombination signal from chromosome 11 and the downstream recombination signal of the TCR D delta 2 gene segment from chromosome 14. In contrast, the 14q- junctions from both patients were diversified by random loss and addition of nucleotides at the translocation site. This asymmetric pattern of junctional diversification is typical of normal Ig/TCR gene rearrangement, and therefore implies that the t(11;14)(p13;q11) translocation arose due to aberrant activity of the Ig/TCR recombinase.     

伴t(16;21)(p11;q22)的恶性血液病的临床和实验分析

目的探讨伴t(16;21)(p11;q22)的恶性血液病的临床及实验室特征。方法骨髓细胞24 h培养后按常规方法制备染色体,用RHG显带技术进行细胞遗传学分析。结果 1例M2的患者其核型分析结果有t(16;21)(p11;q22)的异常,临床和血液学改变符合急性髓细胞白血病-M2a诊断,化疗后未获得完全缓解,中位生存期为6个月。结论 t(16;21)(p11;q22)是一类很独特的白血病亚型有关的易位,为少见的非随机的染色体易位,其临床预后差。     

慢性粒细胞白血病急变伴t(3;21)(q26;q22)染色体易位受累基因的研究

目的探讨慢性粒细胞白血病（CML）急性髓系白血病（AML）变伴t（3;21）（q26;q22）的受累基因.方法对1例CML AML变伴t（3;21）（q26;q22）患者细胞间期和中期分裂相细胞采用荧光原位杂交技术（FISH）检测AML1和bcr/abl基因重排,RT-PCR联合序列分析检测t（3;21）（q26;q22）受累基因.结果der（3）和der（21）染色体上均检测到AML1基因杂交信号,AML1-MDS1-Evi1、AML1-MDS1、AML1-EAP及Evi1基因均表达,未见AML1-Evi1融合基因表达,AML1-MDS1-Evi1基因表达水平是AML1-MDS1、AML1-EAP表达水平的1.58和1.54倍,患者Evi1基因表达水平是HEL细胞系Evi1表达水平的2.71倍.结论t（3;21）（q26;q22）导致形成AML1-MDS1-Evi1、AML1-MDS1融合基因及Evi1基因激活,这些继发的分子遗传学异常是CML急性变伴t（3;21）（q26;q22）患者急变发生的分子基础.     

Rearrangements of the tal-1 locus as clonal markers for T cell acute lymphoblastic leukemia.

Normal and aberrant immune receptor gene assembly each produce site-specific DNA rearrangements in leukemic lymphoblasts. In either case, these rearrangements provide useful clonal markers for the leukemias in question. In the t(1;14)(p34;q11) translocation associated with T cell acute lymphoblastic leukemia (T-ALL), the breakpoints on chromosome 1 interrupt the tal-1 gene. A site-specific deletion interrupts the same gene in an additional 26% of T-ALL. Thus, nearly one-third of these leukemias contain clustered rearrangements of the tal-1 locus. To test whether these rearrangements can serve as markers for residual disease, we monitored four patients with T-ALL; three of the leukemias contained a deleted (tald) and one a translocated (talt) tal-1 allele. These alleles were recognized by a sensitive amplification/hybridization assay. tald alleles were found in the blood of one patient during the 4th mo of treatment but not thereafter. Using a quantitative assay to measure the fraction of tald alleles in DNA extracts, we estimated that this month 4 sample contained 150 tald copies per 10(6) genome copies. The patient with t(1;14)(p34;q11) (talt) leukemia developed a positive assay during the 20th mo of treatment. By standard criteria, all four patients remain in complete remission 11-20 mo into treatment. We conclude that tal-1 rearrangements provide useful clonal markers for approximately 30% of T-ALLs.     

MYC rearrangement involving a novel non-immunoglobulin chromosomal locus in precursor B-cell acute lymphoblastic leukemia

MYC rearrangement, a characteristic cytogenetic abnormality of Burkitt lymphoma and several subsets of other mature B-cell neoplasms, typically involves an immunoglobulin gene partner. Herein, we describe a case of precursor B-cell lymphoblastic leukemia harboring a MYC rearrangement with a novel non-immunoglobulin partner locus. The patient was a 4-yr-old Korean boy with ALL of the precursor B-cell immunophenotype. At the time of the second relapse, cytogenetic analyses revealed t(4;8)(q31.1;q24.1) as a clonal evolution. The MYC rearrangement was confirmed by FISH analysis. He died 3 months after the second relapse without achieving complete remission. To our knowledge, this is the first report of a case of MYC rearrangement with a non-immunoglobulin partner in precursor B-cell lymphoblastic leukemia.     

T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region.     

Coding sequences of the tal-1 gene are disrupted by chromosome translocation in human T cell leukemia

The tal-1 proto-oncogene encodes a helix-loop-helix DNA-binding protein that has been implicated in the formation of T cell acute lymphoblastic leukemia (T-ALL). Patients with T-ALL harbor structural rearrangements of tal-1 that result from either local DNA deletion or t(1;14)(p34;q11) chromosome translocation. By analyzing t(1;14)(p34;q11) chromosomes from a series of patients, we have now identified a discrete region of tal-1 wherein most of the translocation breakpoints occur. Moreover, mapping of tal-1 genomic DNA revealed that coding exons are situated on both sides of the t(1;14)(p34;q11) major breakpoint region. Hence, the translocated allele of tal-1 is truncated in a manner that reduces its amino acid coding potential.     

二例伴有dic(9;20)(p11-13;q11)急性淋巴细胞白血病患者的临床和实验研究

目的探讨伴有dic（9;20）（p11-13;q11）的急性淋巴细胞白血病（ALL）的细胞形态学、免疫学、细胞遗传学特征和临床特点.方法骨髓细胞经直接法和24h短期培养后按常规方法制备染色体,采用R显带技术进行细胞遗传学分析.分别以9号和20号染色体着丝粒探针进行双色荧光原位杂交（FISH）检测.结果2例患者的临床和血液学改变符合ALL诊断,免疫表型分析B淋系标志阳性（CD10＋、HLA-DR＋）;染色体核型分析显示2例患者均为dic（9;20）：例1为45,XY,der（9）t（9;20）（p11;q11）,-20[20],例2为45,XX,der（9）t（9;20）（p13;q11）,t（9;22）（q34;q11）,-20[10]/46,idem,＋8[16]/47,idem,＋8,＋21[14];其中1例经双色FISH检测证实9号和20号染色体之间发生了相互易位,且形成双着丝粒染色体.结论dic（9;20）（p11-13;q11）是一种少见的重现性核型异常,可能和ALL有特殊的联系.FISH技术是检测该易位的可靠手段.     

CD5-negative blastoid variant mantle cell lymphoma with complex CCND1/IGH and MYC aberrations

The coexistence of CCND1/IGH and MYC rearrangements in mantle cell lymphoma (MCL) is a rare finding associated with a very poor prognosis. In this study, a patient with blastoid variant (MCL) is reported. The disease was clinically aggressive and refractory to chemotherapy, and the patient only survived for 1 month following diagnosis. Conventional cytogenetic study, FISH, and multicolor FISH (mFISH) demonstrated the involvement of the BCL1/CCND1 locus in a complex translocation, t(3;11)(q25;p15)t(11;14)(q13;q32). In addition, subclonal abnormalities in the 8q24 region, manifested as a t(8;14)(q24;q32)/MYC rearrangement, were identified. To the best of our knowledge, this is the first MCL case in Korea bearing these complex genomic aberrations.     

SET-NUP214融合基因在急性T淋巴细胞白血病患者中的表达及临床意义

本研究旨在探讨SET-NUP214融合基因在急性T淋巴细胞白血病(T-ALL)中的表达,分析伴有SET-NUP214的T-ALL的临床及分子生物学特征。运用RT-PCR检测58例T-ALL患者骨髓标本中SET-NUP214的表达,以间期荧光原位杂交(FISH)及微阵列比较基因组杂交(Array-CGH)检测9q34缺失,基因测序法检测PHF6和NOTCH1基因突变。结果表明,仅在6例T-ALL患者中检测到SET-NUP214融合基因的表达。除T系抗原标记外,这些患者均表达髓系抗原CD13和(或)CD33,其中4例患者经FISH检测到9q34缺失,3例患者经Array-CGH检测到del(9)(q34.11q34.33)。6例SET-NUP214阳性的T-ALL患者中有4例检测到PHF6基因突变,5例检测到NOTCH1基因突变。结论:SET-NUP214融合基因多由染色体9q34的缺失所致,SET-NUP214阳性的T-ALL常伴有PHF6及NOTCH1基因突变。     

Detection of translocations diagnostic for Berkitt's lymphoma by fluorescent in situ hybridization on histological sections of paraffin blocks

AIM: To test feasibility of detection of translocations which are diagnostic for Berkitt's lymphoma with the method of fluorescence in situ hibridization (FISH) on histological sections of paraffin blocks. MATERIAL AND METHODS: FISH on histological sections for detection of t(8;14)(q24;q32) and variant t(2;8)(p12;q24) and t(8;22)(q24;q11) was performed on the material obtained from 53 patients with typical clinical, morphological and immunological picture. DNA probe LSI IgH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe (Vysis, USA), for variant translocations DNA-probe LSI MYC, Dual color, Break Apart Rearrangement Probe (Vysis, USA) were used. RESULTS: Histological material from 31 patients contained translocations characteristic for LB: in 29 (93.5%)--t(8;14)(q24;q32), in 2--variant rearrangements of locus of gene c-myc. Translocation t(8;14)(q24;q32) and its variants were not detected in 22 patients, the diagnosis was changed for diffuse large B-cell lymphoma (DLBCL). CONCLUSION: Typical for BL clinical, morphological and immunological picture may present in extra-nodal diffuse large B-cell lymphoma with high proliferative activity. Differential diagnosis between BL and the latter lymphoma is possible only basing on detection of translocation t(8;14)(q24;q32) or its variants. If it is impossible to obtain native material, FISH on histological sections of parasffin blocks is the only possible method of differential diagnosis.     

8p11骨髓增殖综合征的临床病理学特征

本研究旨在提高对罕见的8p11骨髓增殖综合征(eight p11myeloproliferative syndrome,EMS)的临床病理特征、诊断与治疗的认识。应用骨髓细胞涂片、骨髓活检观察细胞形态学改变,流式细胞术检测骨髓细胞的免疫表型,细胞遗传学方法分析骨髓细胞核型,分子生物学检测bcr/abl融合基因。结果表明:EMS是一组具有独特临床和生物学特点的疾病,以bcr/abl阴性的骨髓增殖性肿瘤合并淋巴母细胞淋巴瘤(lymphoblastic lymphoma,LBL)为主要特征。骨髓细胞形态学提示粒系细胞高度增生、嗜酸粒细胞增多;免疫表型分析显示髓系抗原表达增高;细胞遗传学分析表明存在8p11易位;RT-PCR检测bcr/abl融合基因呈阴性。在分子学水平,患者染色体异常均累及8p11上的成纤维细胞生长因子受体-1(FGFR1),目前共发现11种与FGFR1重排相关的伙伴基因,其中最常见的是位于13q11-12上的ZNF-198。EMS预后不良,患者常在短期内进展为急性髓系白血病,常规化疗效果差,除异基因造血干细胞移植外无有效的治疗方法。结论:EMS是伴有FGFR1重排的骨髓和淋巴肿瘤,易误诊为T-LBL、不典型慢性粒细胞白血病(aCML)及慢性粒-单核细胞白血病(CMML),对该病应及时进行细胞遗传学及分子生物学检测,以避免误诊、误治。