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。     

Acute leukemia with B-lymphoid and myeloid differentiation associated with an inv(5)(q13q33) in an adult patient

A paracentric inversion in the long arm of chromosome 5, inv(5)(q13q33), was found as a clonal abnormality in a patient with a treatment-resistant biphenotypic acute leukemia with B-lymphoid and myeloid differentiation. This cytogenetic aberration has not been reported previously in biphenotypic acute leukemia, although rearrangements of chromosome bands q13 and q33 have been implicated in the development of several hematologic malignancies. The presence of this aberration as the sole chromosomal abnormality suggests an important role in the pathogenesis of biphenotypic acute leukemia.     

29例伴del(20q)骨髓增生异常综合征的细胞遗传学和临床特征

目的 分析伴del(20q)的骨髓增生异常综合征(myelodysplastic syndrome，MDS)患者的细胞遗传学和临床特征。方法 对29例伴del(20q)MDS的细胞遗传学改变、临床表现、实验室检查特点及病程转归进行总结分析。结果 (1)29例del(20q)的MDS中，11例(37．9％)混合正常核型，难治性贫血(refractory anemia，RA)／伴环形铁粒幼细胞增多的难治性贫血(RA with ringed sideroblasts，RAS)组有9例，而原始细胞增多的RA(RA with excess blasts，RAEB)／转变中的RAEB(RAEB in transformation，RAEB-T)组2例；RA／RAS组中缺失以del(20q)(q11)多见(63．2%)，而RAEB／RAEB-T组中以del(20q)(q12)多见(70．0％)；RA／RAS组的附加核型改变和复杂核型改变发生率为26．3％、5．3％，均低于RAEB／RAEB-T组的50．0％和30．0％；(2)伴del(20q)的MDS多表现为两系或3系血细胞减少，几乎全部患者有红系和粒系病态造血，而58．6%的患者有巨核细胞病态造血，13例(44．8％)患者为两系病态造血，14例(48．3％)患者为3系病态造血，另2例为单纯红系病态造血；62．5％患者的有核红细胞糖原染色阳性和中性粒细胞碱性磷酸酶积分减低；81．8％患者有淋巴细胞的免疫学标记表达；(3)2例患者转化为急性非淋巴细胞性白血病(acute nonlymphocytic leukemia，ANLL)M2a型。结论 del(20q)可能是血液肿瘤中一种早期和初步的细胞遗传学异常，伴del(20q)的MDS以两系和3系血细胞减少及病态造血常见，可表达淋巴细胞标记，随病情进展正常核型减少而附加和复杂核型增加。     

A novel subtelomeric translocation t(5;9) and a deletion of the RB1 gene in a patient with acute myeloid leukemia (AML-M0)

No chromosomal rearrangements have been identified as specifically associated with minimally differentiated acute myeloid leukemia (AML-M0). Several research groups studied the cytogenetic features of AML-M0 and found that as much as 81% of patients with AML-M0 had chromosomal rearrangements; primarily -5/5q- and/or -7/7q- deletions or translocations involving 12p. A patient, who was diagnosed with AML-M0 eighteen months ago, was referred for cytogenetic evaluation for possible AML relapse. A subtle, cryptic t(5;9)(q35.3;q34.3), plus a deletion of the RB1 gene were detected in 18 out of 20 cells analyzed by FISH utilizing the TelVysion assay kit. To rule out the possibility that these chromosomal changes were related to the relapse of AML in this case, we repeated the same FISH test on the specimen at initial diagnosis before any treatment. The same abnormalities were found. To our knowledge, this is the first case reported with subtelomeric t(5;9)(q35.3;q34.3) and the deletion of the RB1 gene in a patient with AML-M0. Whether the t(5;9) combined with the deletion of the RB1 gene plays an important role in the development of AML-M0 warrants further investigation.     

Fluorescence in situ hybridization confirmation of 5q deletions in patients with hematological malignancies

Fluorescence in situ hybridization (FISH) using specific probes for the 5q31-32 region and a whole chromosomal painting (WCP) probe for chromosome 5 were used to corroborate the results of classical cytogenetic examinations performed on G-banded chromosomes of 77 patients with hematological malignancies. Using classical cytogenetic methods, we suspected the presence of clones with a deletion 5q in 63 patients, and complex rearrangements with involvement of chromosome 5 in 14 other cases. Fluorescence in situ hybridization proved the occurrence of deletion 5q31 in 23 patients and ascertained translocations of part of the long arms of deleted chromosome 5 with missing region 5q31 in 12 patients. In 2 cases, the 5q31 region was translocated to other chromosomes as a part of complex rearrangements. The combination of classical cytogenetics and FISH with specific probes for the 5q31 band yielded cytogenetic results in 35 cases. Routine FISH detection of deleted regions was possible by commercially available cosmid probes for the 5q31 chromosomal band. The interpretation of small deletions and frequent involvement of the deleted chromosomes 5 in complex translocations were ascertained by WCP probes.     

Spectra of chromosomal aberrations in 325 leukemia patients and implications for the development of new molecular detection systems

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.     

Chromosome 16 abnormalities associated with myeloid malignancies

Twenty-six patients who had cytogenetic analyses performed for myeloid malignancies at St. Vincent's Hospital over a 6-year period were found to have an inversion abnormality of chromosome 16 (25 patients) or t(16;16) (1 patient). Only 16 patients had all the features of M4Eo, while the other 10 patients had diagnoses of M2, M4, M5, RAEB, and RAEB-T; six of these had abnormal eosinophils. Thus, abnormal eosinophils were present in 22 of 26 patients (85%). Thirteen patients had additional cytogenetic abnormalities at diagnosis, the commonest being +8 in 5, del(7q) in 4, and +21 in 3. Twenty-three patients received chemotherapy and 20 (87%) achieved complete remission. The median survival of the treated group was 188 weeks with a 61% 2-year and 45% 5-year survival. No significant difference in survival was observed between those patients with a diagnosis of M4Eo and those with other diagnoses suggesting that it is the abnormality of chromosome 16 which confers an improved prognosis. Additional cytogenetic abnormalities present at diagnosis did not affect prognosis. CNS relapse was observed in only two patients (8%), thus indicating no increased incidence of this complication. This study supports the premise that a chromosome abnormality involving 16p13 and 16q22 defines a good prognosis subset of myeloid leukemia despite morphological variations.     

联合间期和中期荧光原位杂交检测发现11q23异常伴D13S319缺失急性髓系白血病一例

目的对1例出现11q23异常伴D13S319缺失的罕见急性髓系白血病(acute myeloid leukemia,AML)患者进行多途径细胞遗传学分析,为诊断、治疗及预后分层提供依据。方法应用G+R显带技术对患者24 h培养后的中期分裂相进行染色体核型分析,联合分裂间期和中期荧光原位杂交(fluorescence in situ hybridization,FISH)技术对患者染色体的特定位点进行检测,明确复杂易位和微小缺失片段。结果患者存在混合系白血病基因(mixed lineage leukemia,MLL)重排,形成了MLL-AF10融合基因,并伴有13号染色体D13S319位点的缺失。结论MLL基因重排合并D13S319位点缺失在急性白血病中是否具有双重打击效应应引起临床的重视。在AML患者核型中发现13q-、del(13)(q14)、-13或der(13)任意一种克隆性异常时,应行FISH检测论证及明确缺失片段的大小,以便进行靶向治疗。     

Is fluorescence in situ hybridization a useful method in diagnosis of polycythemia vera patients?

Polycythemia vera (PV) is a clonal stem cell disease with trilineage myeloid involvement, characterized by a growth factor-independent erythroid proliferation. At the time of diagnosis, the percentage of cytogenetic abnormalities using conventional cytogenetic techniques is less than 20% of all PV patients. In the present study, we compare the results between conventional cytogenetic methods and fluorescence in situ hybridization (FISH) probes in 31 untreated PV patients. The karyotypes of all 31 cases were obtained from 24-hour bone marrow cell cultures. The fixed material, proceeding from conventional cytogenetics cultures, was analyzed with FISH, using centromeric probes for chromosomes 8 and 9 and locus-specific probes for 13q14 and 20q12 regions. Five cases (17.8%) showed an abnormal karyotype with conventional cytogenetics. When FISH probes were used, three alterations not detected with conventional cytogenetics, were found: two cases with D20S108 deletions and one with a D13S319 deletion, increasing the percentage of abnormal karyotypes to 19.3%. We conclude that, probably, the application of FISH with the mentioned probes, is not very useful to detect cytogenetic aberrations in untreated PV patients.     

MLL amplification in myeloid leukemias: A study of 14 cases with multiple copies of 11q23

We here report the clinical, cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data on 14 patients with a myeloid malignancy and structural aberration of chromosome band 11q23 associated with overrepresentation or amplification of the MLL gene. The number of copies of MLL varied from three (two cases) to a cluster consisting of multiple hybridization spots. Together with previous reports, available data indicate that amplification of 11q23/MLL is a recurrent genetic change in myeloid malignancy. It affects mainly elderly patients and is often associated with dysplastic bone marrow changes or with complex karyotypic aberrations, suggestive of genotoxic exposure. It is associated with a poor prognosis. In addition, FISH analysis of nine cases with additional 11q probes showed that the overrepresented chromosomal region is generally not restricted to MLL, and Southern blot analysis indicated that amplification does not involve a rearranged copy of this gene. The significance of MLL amplification and the mechanisms by which it could play a role in leukemogenesis and/or disease progression remain to be elucidated.     

Frequency, hematopathology, and detection of a new isodicentric variant of deletion 20q

The ider(20)(p11.21)del(20)(q11q13) anomaly was recognized only recently. Thus, its frequency and clinical significance has not been extensively studied. Due to small size and ambiguous G-band pattern, ider(20q) is usually missed in cytogenetic studies. Furthermore, the commercial FISH probe D20S108 does not distinguish among del(20q), ider(20q), and monosomy 20. Thus, we determined the frequency and hematopathology of patients with ider(20q), and the best cytogenetic methods to detect chromosome 20 anomalies. To do this, we performed FISH on interphase and metaphase cells for 12 patients with -20,+mar and 12 patients with only del(20q) in their karyotype. The marker chromosome in patients with -20,+mar proved to be ider(20q). FISH with D20S108 and 20qter distinguished ider(20q) from del(20q) and monosomy 20. Review of blood and bone marrow slides for nine patients with ider(20q) showed that one had acute myeloid leukemia and eight had myelodysplastic syndromes. Patients with ider(20q) had a more consistent presentation of multilineage dysplasia with additional involvement of the granulocytic series than patients with del(20q). This study shows ider(20q) is common in clinical practice--1/10th the incidence of del(20q)--and is strongly associated with myelodysplasia and acute myeloid leukemia.     

综合应用分子细胞遗传学技术检测一例染色体微小易位

目的 综合应用分子细胞遗传学技术对1例染色体微小易位的病例进行检测.方法 按常规制备染色体,G显带进行核型分析,并先后进行光谱核型分析(spectral karyotyping,SKY),染色体涂染,双色荧光原位杂交技术(fluorescence in situ hybridisation,FISH)检测,亚端粒探针F...     

Interphase fluorescence in situ hybridization assay for the detection of 3q21 rearrangements in myeloid malignancies.

In myeloid malignancies, chromosome rearrangements involving band 3q21 are associated with a particularly poor prognosis of the disease. Their sensitive and unequivocal detection is therefore of great clinical importance. In this report, we describe the establishment of an interphase fluorescence in situ hybridization (FISH) assay that complements classical cytogenetic analysis in the diagnosis of such aberrations. PACs that map centromeric and telomeric of known 3q21 breakpoints were labeled with different fluorescent dyes, and the separation of the normally colocalizing signals was used as an indicator of the presence of a 3q21 rearrangement. Two cell lines and 10 primary samples from myeloid leukemia and myelodysplastic syndrome (MDS) patients with 3q21 rearrangements were investigated using the newly established method. The rate of false positivity was determined in 27 control samples from patients with various types of myeloid malignancies. In addition to providing a sensitive and rapid test for the detection of 3q21 aberrations, the interphase FISH assay yields preliminary information about the localization of individual breakpoints. Six of the 10 breakpoints in the patient samples map to an only recently described breakpoint cluster region (BCR) 60 kb centromeric of the originally reported 3q21 BCR. These findings may contribute to the understanding of the molecular basis of the clinical features associated with 3q21 rearrangements.     

Isochromosome (17)(q10) and translocation (4;12)(q12;p13) in a child with acute myeloid leukemia.

Isochromosome 17q is a commonly observed cytogenetic aberration in hematologic malignancies. Isolated isochromosome 17q usually presents as a marker of a chronic myeloid disorder, with a high propensity for transformation into acute nonlymphoblastic leukemia (ANLL). t(4;12)(q11-12;p13) is a recently described translocation, associated with ANLL, predominantly in adults. In this article, we present a case of acute myeloblastic leukemia (AML) in a 14-year-old female in which i(17q) and t(4;12)(q12;p13) were found in the leukemic clone at diagnosis. We briefly review the literature and hypothesize as to the significance of the coexistence of these cytogenetic changes.     

The human Penumbra gene is mapped to a region on chromosome 7 frequently deleted in myeloid malignancies

We previously cloned the murine Penumbra gene based on its differential expression in proerythroblasts/erythroblasts. Subsequently, we identified human Penumbra cDNA from a human bone marrow cDNA library and the human Penumbra gene from a BAC library. Penumbra is a new member of the tetraspanin protein family and exhibits growth-suppressive activity in vitro. In this study, we designed a human Penumbra probe contig and used fluorescent in situ hybridization (FISH) to analyze seven cases of myeloid malignancies with 7q deletions. Five patients with cytogenetic deletions involving 7q31.2 approximately q32 also showed deletions of Penumbra by FISH; these were not present in two patients with cytogenetic deletions not involving 7q31.2 approximately q32. Our findings provide the first FISH evidence supporting the mapping of human Penumbra to 7q31.2 approximately q32 and demonstrate the potential of the Penumbra probe in the detection of 7q31 approximately q32-related deletions in myeloid malignancies.     

Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients

Abstract Myelodysplastic syndrome (MDS) involves myeloid cells of the bone marrow, which is important in progressive bone marrow insufficiency. Of all MDS patients, 40%–50% have at least one chromosomal rearrangement. Loss of specific chromosomal regions like 5q– and 7q– are usually the secondary cytogenetic abnormalities associated with MDS. In order to detect chromosome abnormalities associated with MDS, bone marrow samples from 26 patients diagnosed as MDS were obtained prior to chemotherapy. Both conventional cytogenetic analyses and fluorescence in situ hybridisation (FISH) methods were performed and locus–specific probes for 5q and 7q were used. Results obtained were compared. Twenty–one patients had normal karyotypes and four patients had abnormal karyotypes, while in one patient we could not obtain metaphases from cultures. Three patients with normal karyotypes revealed del (5q), two patients had del (7q) and one patient had monosomy (7). A total of 10 of 26 patients had chromosome changes visualised by either conventional or molecular cytogenetics (~38.5%). Our results show that both methods are important in diagnosis and follow up of MDS patients. When used together, conventional cytogenetics and FISH detect clinically significant chromosome abnormalities in MDS patients.     

Apparently unrelated cytogenetic abnormalities among 462 probands referred for the detection of del(22q) by FISH

Laboratory-based reports of the cytogenetic abnormalities detected during the course of testing for deletion del(22q) are scant. We report our findings from the testing with FISH of 462 patients suspected to have del(22q) between 1994 and 2000. Of these, 447 had a normal karyotype. An apparently unrelated cytogenetic abnormality was detected in 15 (3.2%). Two of these abnormalities involved reciprocal translocation with chromosome 22q and one of these showed del(22q) with FISH. The other abnormalities included sex chromosome aneuploidies and unbalanced rearrangements of various chromosomal segments. There was no commonality among these abnormalities and no correlation with other reported cases. Among those with a normal karyotype, an unexpected deletion of the control arylsulphatase A (ARSA) probe was found, providing a definite frequency of 1/262 for ARSA deletions among patients suspected to have del(22q). Of the 462 referrals, 48 (10%) had one or more additional diagnoses, and in this group, 4 (8%) had del(22q) and 2 (4%) had an apparently unrelated cytogenetic abnormality. The data highlight the importance of initial cytogenetic analysis in patients suspected of del(22q) and negates the use of interphase FISH screening by itself for del(22q). The finding of 3.2% unrelated cytogenetic abnormalities is noteworthy. FISH should be used in any structural rearrangement to ascertain if the relevant locus is deleted or not. The continued reporting of patients diagnosed with del(22q) found to have an unrelated cytogenetic abnormality will expand the phenotypic spectrum and possible gene mapping may refine phenotypic specificity.     

A case of atypical myelodysplastic syndrome with micromegakaryocytes,normal platelet count,and t(3;12)(q21;p13) with inv(3)(q21q26)

A 49-year-old woman patient with atypical myelodysplastic syndrome (MDS) showing a der(3)t(3;12)(q21;p13), and der(12)t(3;12)(q21;p13)inv(3)(q21q26) as an acquired chromosomal abnormality in the bone marrow is described. The chromosomal breakpoints of the presented complex aberration with combination of the inv(3)(q21q26) and t(3;12)(q21;p13) were defined by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs). The inv(3) is a relatively frequent chromosomal rearrangement in patients with myeloid malignancies and dysmegakaryopoiesis and t(3;12)(q26;p13) has also been reported as a recurrent abnormality in MDS and in blast crisis of chronic myelogenous leukemia (CML). Whereas the t(3;12), inv(3), and t(3;3) are associated with a very poor prognosis, our patient surprisingly had a mild clinical course. Genes Chromosomes Cancer 20:292–298, 1997. © 1997 Wiley-Liss, Inc.     

Novel chromosomal aberrations in a recurrent malignant meningioma

The molecular basis of tumorigenesis and tumor progression in meningiomas is not fully understood. Here we present results of conventional cytogenetic, fluorescence in situ hybridization (FISH), and comparative genetic hybridization (CGH) analyses in a patient with recurrent anaplastic meningioma. We found complex aberrant karyotype alterations previously described in anaplastic meningiomas, such as 1p, 14q aberration, and a possibly tetraploid karyotype. Loss of chromosome 22q was detected by conventional cytogenetic analysis. Additional chromosomal aberrations not previously reported included a near-triploid karyotype and alterations such as 4p+, 5p-, 7p+, 8q+, and gain of chromosome 19. FISH with LSI 9p21, CEP9, LSI PML/RARA, and CGH confirmed the karyotype complexity in this case. Our findings of several previously unreported cytogenetic alterations suggest that complex karyotype alterations are a characteristic feature in anaplastic meningiomas. High chromosomal complexity might be associated with a highly aggressive meningioma phenotype.     

Incidence of submicroscopic deletions vary according to disease entities and chromosomal translocations in hematologic malignancies: investigation by fluorescence in situ hybridization

Submicroscopic deletions of genes in recurrent chromosomal rearrangements occur frequently in hematologic malignancies, but their incidences have not been reported clearly. We investigated the incidence of submicroscopic deletions and their association with specific genetic rearrangements in various hematologic malignancies. A fluorescence in situ hybridization (FISH) study was conducted in 336 patients with acute lymphoblastic leukemia, 223 patients with acute myeloid leukemia, and 79 patients with chronic myelogenous leukemia. The incidence of submicroscopic deletions in patients with chromosomal rearrangements was the highest in the TEL/AML1 rearrangement (65.0%), followed by BCR/ABL (10.9%), MLL (5.6%), AML/ETO (4.0%), and PML/RARA (0.0%). Submicroscopic deletion was quite common, and incidences were variable according to disease entities and chromosomal translocations. To detect submicroscopic deletions, careful FISH study should be included for the cytogenetic study of hematologic malignancies, and their association with clinical prognosis needs to be further studied.