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.     

套细胞淋巴瘤分子遗传学研究进展

套细胞淋巴瘤(mantle cel l lymphoma,MCL)是一种具有独特生物学特征的非霍奇金B细胞淋巴瘤(non-Hodgkin l ymphoma,NHL),其特征是标志性易位t(11;14)(q13;q32)和细胞周期蛋白D1(cyclinD1)的过度表达。MCL的分子遗传学改变包括频繁的继发性细胞遗传学异常和少数基因的反复突变,如CCND1,SOX11等。本文对MCL近年来在分子遗传学方面的研究进展,包括细胞循环周期失调、DNA损伤应答改变以及其他相关基因的异常改变作一综述。     

Fluorescence in situ hybridization analysis of immunoglobulin heavy chain translocations in plasma cell myeloma using intact paraffin sections and simultaneous CD138 immunofluorescence

Fluorescence in situ hybridization (FISH) studies are much more sensitive than classical cytogenetics for identification of karyotypic abnormalities in plasma cell myeloma. However, FISH analysis of bone marrow samples is often challenging because of a large number of admixed non-neoplastic hematopoietic elements. In this report, we describe a novel method using FISH analysis of intact paraffin sections of formalin-fixed, bone marrow clot preparations with simultaneous CD138 tyramine signal amplification (TSA)-mediated immunofluorescence. We studied 22 cases of plasma cell myeloma for translocations involving the immunoglobulin heavy chain locus that are of known diagnostic and/or prognostic significance. All cases were analyzed using dual color, break-apart immunoglobulin heavy chain probe and dual color, dual fusion probes for t(11;14)(q13;q32) and t(4;14)(p16;q32). TSA-mediated fluorochrome deposition in CD138+ cells was unaltered by protease pretreatment. Translocations were identified in 10 cases, including five with t(11;14)(q13;q32) and three with t(4;14)(p16.3;q32). When present, abnormalities were identified in a large percentage of CD138+ cells (47 to 93%, median 84%). This technique allows for efficient molecular cytogenetic analysis of plasma cell myeloma using routinely archived paraffin-embedded material.     

Simultaneous translocation of both TCR Loci (14q11) with rare partner loci (Xq22 and 12p13) in a case of T-lymphoblastic leukemia

The most common recurrent cytogenetic abnormalities in T-lymphoblastic leukemia (T-acute lymphoblastic leukemia [T-ALL]) involve T-cell receptor (TCR) loci and a variety of partner genes, including HOX11, HOX11L2, MYC, and TAL1. In this report, we present a rare case involving simultaneous translocation of the TCR α/δ loci with different partner loci (Xq22 and 12p13); this resulted in a poor prognosis. Chromosomal analysis showed 46,Y,t(X;14)(q22;q11.2),t(12;14)(p13;q11.2) and FISH analysis by using a T-cell receptor alpha delta DNA probe, Split Signal (DakoCytomation, Denmark), showed translocations at the same TCR α/δ locus on both chromosomes. FISH with 2 bacterial artificial chromosome clones showed break apart signal, which suggests involvement of the IRS4 gene. To our knowledge, this is the first report of T-ALL in which both TCR α/δ loci were translocated with different partner loci, and 1 of the partner loci, Xq22, was a rare translocation partner locus that included IRS4 gene.     

初治多发性骨髓瘤患者的基因突变及表达异常

目的:分析初治多发性骨髓瘤(NDMM)患者常见基因的突变及表达异常。方法:取208例NDMM患者的骨髓细胞,用DNA测序法检测6种基因的表达水平及28种基因的突变状态。应用FISH法检测多发性骨髓瘤常见的细胞遗传学异常。结果:61例(29.33%)NDMM患者检测到基因突变,≥5例患者发生突变的基因包括NRAS、PRDM1、FAM46C、MYC、CCND1、LTB、DIS3、KRAS和CRBN。83例(39.90%)患者检测到6种基因的过表达,以细胞周期调节基因的过表达为主,分别是CCND1、CCND3、BCL-2、CCND2 FGFR3和MYC。169例(81.25%)患者检测到基因单核苷酸多态性改变,主要为TP53基因P72R多态性(70.17%)。染色体结构异常与基因表达异常有一定相关性,与染色体结构正常者相比较,14q32缺失患者中CCND1基因过表达比例更高,13q14缺失患者中FGFR3过表达比例更高,而1q21扩增患者中CCND2、BCL-2、FGFR3基因过表达比例更高。结论:NDMM患者存在多种基因突变和表达异常,但是缺乏主要的单个基因突变或表达异常。累及RAS/MA...     

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.     

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma

In mantle cell lymphoma (MCL) and some cases of multiple myeloma (MM), cyclin D1 expression is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus. To evaluate the mechanisms responsible, gene targeting was used to study long-distance gene regulation. Remarkably, these targeted cell lines lost the translocated chromosome (t(11;14)). In these MCL and MM cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome induced a loss of methylation at the unrearranged CCND1 locus, providing evidence of a transallelic regulatory effect. In these cell lines and primary MCL patient samples, the CCND1 loci are packaged in chromatin-containing CCCTC binding factor (CTCF) and nucleophosmin (NPM) at the nucleolus. We show that CTCF and NPM are bound at the IgH 3′ regulatory elements only in the t(11;14) MCL cell lines. Furthermore, NPM short hairpin RNA produces a specific growth arrest in these cells. Our data demonstrate transvection in human cancer and suggest a functional role for CTCF and NPM.     

人多发性骨髓瘤细胞系与初治多发性骨髓瘤遗传学异常的比较研究

本研究采用荧光原位杂交（fluorescence in situ hybridization,FISH）检测人多发性骨髓瘤细胞系（human multiple myeloma cell lines,HMCL）和初治多发性骨髓瘤（MM）的分子遗传学异常并进行比较分析,以帮助筛选浆细胞肿瘤高危遗传学异常,为多发性骨髓瘤MM的危险度分层提供依据,并为利用HMCL进行MM研究提供遗传学资料。选用13q14（RB-1）、14q32（IGHC/IGHV）、1q12（CEP1）、17p13（TP53）荧光探针应用直接法检测7株HMCL,用CD138免疫磁珠分选（magnetic-activated cell sorting,MACS）联合FISH方法检测85例初治MM患者作为对照。对于存在14q32异常的患者进行IGH/CCND1、IGH/FGFR3、IGH/MAF双色双融合探针杂交以检测t（11;14）（q13;q32）、t（4;14）（p16;q32）、（14;16）（q32;q23）。结果显示：7株HMCL中RB-1/D13S19缺失6株（85.7%）,P53缺失5株（71%）,1q21扩增6株（85.7%）;5株（71.4%）HMCL存在IGH异常,IGH/CCND1阳性细胞系1株,IGH/FGFR3阳性细胞系2株,IGH/MAF阳性细胞系3株;其中KMS11细胞系同时存在IGH/FGFR3和IGH/MAF两种异常。85例初治MM细胞遗传学总体检出率为85.9%,38例（44.7%）伴有RB-1缺失,17例（20%）出现p53缺失,45例（52.9%）伴有1q21扩增,53例（62.4%）存在IGH异常,其中IGH/CCND1阳性23例（27.1%）,IGH/FGFR3阳性21例（24.7%）,IGH/MAF阳性3例（3.5%）。与初治MM相比较,HMCL的抑癌基因p53缺失率和IGH/MAF明显升高（p=0.008,p=0.005）,而其他分子遗传学异常检出率未达统计学差异。结论：HMCL作为浆细胞肿瘤恶性程度最高的形式,积累了大量的遗传学异常,p53缺失是其显著特征。绝大多数HMCL来源于疾病晚期的髓外浆细胞肿瘤细胞或者浆细胞白血病,提示p53缺失是这部分极高危浆细胞肿瘤患者的特征。     

伴有14q32异常的淋巴系统恶性肿瘤的临床与细胞遗传学分析

目的 分析14q32异常的淋巴系统恶性肿瘤（lymphiod maligances,LM)的临床及细胞遗传学特征。方法 应用常规细胞遗传学技术对225例不同类型的LM进行研究并综合分析伴14q32异常LM的临床及实验室特征。结果 15（6.67%）LM患者可见14q32异常,不同类型的14q32异常的分布与LM的不同类型有关。t（8;14）（q24;q32)）最为常见,主要见于急性白血病,其白血病类型呈现形态学及免疫表型特征的异质性,但仍具有独特的临床、预后特征和附加的染色体异常ins（1;6）(q11;q23q27)。t(11;14）（q13;q32)仅见于浆细胞白血病,1例多发性骨髓瘤继发骨髓增生异常综合征（MDS）患者可见累及MDS常见的染色体异常7q-和20q-。结论 特征性14q32异常与附加染色体改变有助于同类型的LM的诊断和预后估计。     

荧光原位杂交技术在套细胞淋巴瘤石蜡切片的应用研究

目的建立淋巴瘤石蜡切片应用荧光原位杂交(fluorescence in situ hybridization,FISH)检测的操作流程,探讨其在套细胞淋巴瘤(mantle cell lymphoma,MCL)诊断中的应用价值。方法优化石蜡切片FISH检测步骤和实验条件,在16例病理拟诊断MCL的石蜡切片上应用FISH检测IGH/CCND1融合基因。结果确立了本实验室石蜡切片FISH检测的实验方法和条件;拟诊断MCL的病例中IGH/CCND1融合基因阳性检出率为75%(12/16)。结论淋巴瘤石蜡切片FISH检测可满足临床应用要求;应用FISH技术检测IGH/CCND1融合基因有助于MCL的诊断。     

荧光原位杂交技术在慢性淋巴细胞白血病检测中的应用评估

本研究探讨与评估应用间期荧光原位杂交技术(FISH)检测慢性淋巴细胞白血病(CLL)遗传学异常的价值。应用间期FISH技术检测32例初诊CLL患者的del(13q14.3)、del(11q22.3)、del(17p13.1)、del(13q14)和12号染色体三体,同时对免疫表型不典型的10例初诊患者检测IGH/CCND1融合基因。结果表明,在32例病例组中FISH检测出26例(81.3%)基因异常,包括D13S25缺失14例,RB1缺失11例,12号染色体三体9例,P53缺失6例,ATM缺失4例;涉及1种基因异常的12例,其中12号染色体三体7例,D13S25缺失3例,P53缺失1例,ATM缺失1例;涉及2种基因异常的11例,其中D13S25/RB1缺失的7例,另4例均包含P53缺失;涉及3种以上基因异常的病例3例;10例免疫表型表达CD5+CD23-的初诊患者中2例IGH/CCND1(+)。结论:应用间期FISH技术检测CLL基因组的异常,可大大提高异常染色体的检出率,各基因异常有其不同的特点;IGH/CCND1融合基因的检测在CLL诊断中有重要意义。     

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.     

套细胞淋巴瘤发病机制的研究进展

套细胞淋巴瘤(MCL)是一种侵袭性的非霍奇金淋巴瘤(NHL),该病的经典遗传学标志为t(11;14)(q13;q32)移位和细胞周期蛋白(cyclin) D1过表达,以难治和易复发为临床特征,并且总体预后不良.近年,随着对MCL细胞遗传学及分子发病机制研究的不断深入,靶向药物的开发与应用,使得该病的临床疗效有所提高.本文就近年来MCL发病机制中细胞遗传学、信号通路、转录因子、肿瘤微环境等改变的研究最新进展作一综述,旨在为MCL的早期诊断和靶向治疗提供新的方向.     

Copy number gains in 11q13 and 8q24 [corrected] are highly linked to prognosis in cutaneous malignant melanoma

Relating specific genetic alterations to prognosis may help improve prognostication in melanoma, may identify key oncogenic drivers in cancer, and may assist in developing targeted therapies. Characteristic genetic alterations in melanoma include chromosomal copy number aberrations. We evaluated 97 melanomas (55 metastasizing and 42 nonmetastasizing) after a minimum 5-year follow-up in a case-control study using fluorescence in situ hybridization, targeting commonly altered chromosomal loci in melanoma. Eight probes arranged in two panels were used, and 11 parameters were evaluated. Parameters showing a statistically significant difference between the metastasizing and nonmetastasizing groups were evaluated with multivariate logistic regression analysis to compare their prognostic potential with other traditional prognostic markers used by the American Joint Committee on Cancer. Four of 11 parameters evaluated, including CCND1 (alias Bcl-1) gain, CCND1 r-gain, MYC (alias c-myc) gain, and MYC r-gain, had a statistically significant difference in the metastasizing versus nonmetastasizing group. All four parameters maintained statistical significance when evaluated in separate multivariate logistic regression analyses that included the seven currently used American Joint Commission on Cancer prognosticators in melanoma. In multivariate analyses, these four parameters were second only to ulceration in their prognostic potential. Copy number changes at 11q13 and 8q24 [corrected] harboring CCND1 and MYC, respectively, are highly associated with prognosis. Fluorescence in situ hybridization targeting these loci may be a useful standardized prognostic marker in melanoma skin cancer.     

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.     

Cytogenetics of mantle cell lymphoma

AIM: To determine the type and rate of secondary chromosomal aberrations and role in pathogenesis of mantle cell lymphoma (MCL). MATERIAL AND METHODS: Standard cytogenetic examination (SCE) and fluorescent in situ hybridization (FISH) with tests for 11q22/ATM, 13q14, 17p13/p53, 9p21/p16 and chromosome 12 centromere were made in 28 patients. RESULTS: Secondary chromosomal aberrations were detected in 22 patients. Chromosomal abnormalities occurring in more than 2 cases include deletions 6q15-q23 (53% cases); 11q22/ATM (50%); 9p21 (36%, in half the cases deletion 9p21 was biallele); 13q14 (32%); trisomy 3/3q, monosomy 20 and deletions/translocations 1q and 1p (27% and 20%) and deletion 17p13/p53 (18%). Trisomies 12/12q, 6 and 18/18q, monosomies 2 and 16, deletions/translocations 2p and 16p were found in 2 cases each. The FISH technique identified 9 chromosomal anomalies missed at SCE. Deletions 6q15-q23 were seen in patients with privalent lesions of lymph nodes. Deletions 11q, 13q14, 9p21 and 17p13 occur more frequently in transformation and the blastoid variant. Monosomy 20 was found only in patients with large cell transformations. This was the only cytogenetic defect characteristic for transformed cases, in contrast to denovoblastoid ones. Thus, deletions 6q, 11q23, 13q14 and 9p21 are typical for MCL. Deletions 9p21 and 17p13 are more characteristic for large cell variants of the tumor. Deletion 17p13 occurs at the terminal stage of the disease in rapidly growing tumor mass and resistance to chemotherapy. FISH technique is effective in detection of submicroscopic rearrangements especially small deletions. No significant differences were found between transformed and de novo blastoid cases. This shows that the blastoid variant is not a specific biological form, it is rather a less typical manifestation of the disease due to some preclinical cytogenetic disorders. CONCLUSION: Progression and transformation of MCL are related to mutation of proapoptotic genes and genes proteins of which are inhibitors of active complexes of D1 cycline. Specification of these mechanisms requires further investigations in patients with different clinicomorphological forms of the disease at various stages of the disease.     

系统性轻链型淀粉样变性细胞遗传学异常检测分析

目的研究系统性轻链型(AL)淀粉样变性患者的细胞遗传学特征。方法收集初诊系统性AL淀粉样变性患者24例和多发性骨髓瘤(MM)患者135例,通过CD138磁珠分选(MACS)结合间期荧光原位杂交(FISH)技术检测系统性AL淀粉样变性和MM患者的细胞遗传学异常,比较二者细胞遗传学异常差异。分析系统性AL淀粉样变性FISH异常与血清游离轻链(sFLC)、N端脑钠肽前体(NT-proBNP)、B型脑钠肽前体(proBNP)、血清肌钙蛋白(cTnT和cTnI)及器官累及之间的关系。结果系统性AL淀粉样变性细胞遗传学异常阳性率为62.5%,其中14q32异位、t(11;14)和+1q21发生率较高,分别为41.7%、37.5%和29.2%。系统性AL淀粉样变性细胞遗传学异常总阳性率、del(13/13q14)缺失率及并存大于或等于3种遗传学异常的发生率均显著低于MM患者,差异有统计学意义(P0.05),而t(11;14)发生率显著高于MM,差异有统计学意义(P0.05)。系统性AL淀粉样变性细胞遗传学异常与临床相关检测指标及器官累及无显著关系(P0.05)。结论 MACS-FISH可用于检测系统性AL淀粉样变性患者的细胞遗传学异常。系统性AL淀粉样变性患者14q32异位、t(11;14)、+1q21有较高的发生率,t(11;14)发生率显著高于MM患者,而并存大于或等于3种细胞遗传学异常和del(13/13q14)发生率显著低于MM患者。     

嗜酸性粒细胞增多症的细胞遗传学研究

为探讨染色体异常克隆在嗜酸性粒细胞增多症诊断和鉴别诊断中的意义及克隆性嗜酸性粒细胞增多症涉及的染色体异常,收集了65例嗜酸性粒细胞增多患者的骨髓标本,培养24小时,采用G显带进行核型分析。结果表明：65例中9例拟诊为急性髓细胞性白血病-M4Eo检出特异性的染色体异常inv（16）,而其余的56例以嗜酸性粒细胞增多待诊的患者中5例检出染色体异常克隆,检出率为8.9%。根据临床、血液学资料并结合染色体检出结果,5例患者最后分别被诊断为急性髓系白血病伴嗜酸性粒细胞增多、慢性嗜酸性粒细胞白血病、8p11骨髓增殖综合征、慢性髓系白血病急变、急性髓系白血病-M4Eo。检出的染色体异常克隆分别为＋14、t（5;12）（q31;p13）、t（8;9）（p11;q32）、t（9;22）（q34;q11）和inv（16）（p13q22）。结论：在嗜酸性粒细胞增多症的诊断中,染色体的检测是判定克隆性和诊断慢性嗜酸性粒细胞白血病的重要手段,应作为常规的检测。     

8号染色体四体、三体共存的t(15;17)(q22;q12)急性早幼粒细胞白血病

本研究报道首例伴有8号染色体四体(四体8)、8号染色体三体(三体8)异常的t(15；17)急性早幼粒白血病(AML-M3a)，并探讨其形态学、细胞遗传学、分子生物学、免疫学及临床特点。用外周血及骨髓标本直接涂片观察形态学改变；采用骨髓细胞24小时短期培养法制备染色体标本，RHG显带技术进行核型分析；以筑巢式逆转录聚合酶链反应(nested RT—PCR)技术检测PML-RARa融合基因转录本；以间期荧光原位杂交(fluorescence in situ hybridization，FISH)技术检测8号染色体数目异常；以流式细胞术检测免疫表型。结果表明：外周血涂片早幼粒细胞占65％，可见中晚幼粒细胞。骨髓涂片显示有核细胞增生明显活跃，粒系83．6％，其中早幼粒细胞占72．4％，胞浆内可见大量紫红色颗粒。染色体核型分析揭示核型为48，XY， 8， 8，t(15；17)(q22；q12)[16]／47，XY， 8，t(15；17)(q22；q12)[3]／46，XY，t(15；17)(q22；q12)[1]。RT—PCR检测PML-RARa( )。FISH检测显示具有1，2，3，4，5，6个绿色荧光信号细胞的百分比分别为0．5，7，19，55，18和0．5。这不但证实了三体8和四体8克隆的存在．还发现存在一个较小的五体8克隆。白血病细胞免疫表型检测显示CD13(96．2％)、CD33(55．9％)、CYMPO(93．5％)阳性，其余抗原包括淋系抗原在内均为阴性。患者生存期只有10天。结论 本例四体8是t(15；17)的继发性改变，可能是三体8克隆进展的结果。伴有四体8的t(15；17)AML-M3预后差。