A new recurrent translocation, t(5;11)(q35;p15.5), associated with del(5q) in childhood acute myeloid leukemia. The UK Cancer Cytogenetics Group (UKCCG)

Partial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harvey ras-1-related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).     

Spectral karyotyping (SKY) refinement of a complex karyotype with t(20;21) in a Ph-positive CML patient submitted to peripheral blood stem cell transplantation

A patient with a Ph-positive chronic myeloid leukaemia (CML) was submitted to allogeneic peripheral blood stem cell transplantation from an HLA-haploidentical related donor 7 years after the diagnosis. Six months later, he showed a disease relapse while cytogenetic analysis displayed a complex karyotype. To characterise the chromosomal rearrangements spectral karyotype (SKY) analysis was used. This redefined all chromosome rearrangements and revealed a t(20;21)(q11;q22). FISH analysis with a specific probe for the AML1 gene disclosed disruption of this gene which was partially translocated on to the long arm of chromosome 20. It is likely that this rearrangement, unusual for CML, was implicated in the disease evolution towards blastic crisis (BC).     

Del(5q) and MLL amplification in homogeneously staining region in acute myeloblastic leukemia: a recurrent cytogenetic association

We report here a 71 year-old female presenting with acute myeloblastic leukemia (FAB-M1) after treatment of essential thrombocythemia with Vercyte. Conventional cytogenetic techniques showed a complex karyotype, 44,XX,−5,−7,−11,add(11)(q23),−14,+mar,+r. The use of several fluorescent in situ hybridizations (FISH) lead to the identification of these complex rearrangements. The marker was found to be tricentric, with pericentromeric material of chromosome 7 inserted in the short arm of chromosome 5, resulting in monosomy 5q and 7q. The derivative chromosome 11 was dicentric and had subtelomeric sequences of 11p on both ends; several copies of the MLL gene were located in two different regions separated by a centromere of chromosome 11. Twenty-one cases, including ours, of myelodysplastic syndromes and acute myelogenous leukemia with MLL amplification present in hsr or dmin were found in the literature. Most of these patients shared some characteristics: they were old, they had de novo acute myeloid leukemia (AML) with a complex karyotype and a short survival, 90% of them having also a del(5q). Therefore, the simultaneous presence of MLL amplification and del(5q) appears to be a nonrandom association that could be the signature of AML in elderly patients with a poor prognosis.     

Chromosome aberrations in de novo acute myeloid leukemia patients in Kuwait

Cytogenetic analysis was successfully performed at the time of diagnosis in 45 patients with de novo acute myeloid leukemia, including 10 children and 35 adults. In approximately 73% of AML patients (35 patients) clonal chromosome abnormalities were detected at the time of diagnosis. Twelve patients (22.8%) had apparently normal karyotypes. Recurring aberrations found in 22 of patients with abnormal karyotypes included t(15;17)(q22;q11), t(8;21)(q22;q22), inv(16)(p13q22), trisomy 8, monosomy 7 and del(5q). The highest frequency of chromosome changes was observed in AML-M3. The occurrence of the classical cytogenetic abnormalities was not a ubiquitous phenomenon. In 11 patients previously not described miscellaneous clonal chromosomal abnormalities were detected. Clonal chromosomal abnormalities detected in AML have shown correlations between specific recurrent chromosomal abnormalities and clinico-biological characteristics of the patients, therefore have been repeatedly shown to constitute markers of diagnostic and prognostic significance. Moreover, ongoing cytogenetic analysis can identify new nonrandom chromosome aberrations in AML and contribute to the identification of novel genes involved in the development of cancer, which can lead to better understanding of the disease pathogenesis.     

Secondary acute myeloblastic leukemia with t(16;21)(q24;q22) involving the AML1 gene

A t(16;21)(q24;q22) translocation was detected by fluorescence in situ hybridization in a patient with acute myeloblastic leukemia previously treated for malignant lymphoma. While the breakpoint on chromosome 21 was within the AML1 gene as determined by FISH, the gene partner on chromosome 16 could not be identified. Band 16q24 appears to be rearranged in several types of myeloid proliferation and a review of the literature shows that these rearrangements most often occur in secondary leukemia and myelodysplastic syndrome or are part of complex chromosomal rearrangements.     

Secondary acute myeloblastic leukemia with t(16;21)(q24;q22) involving the AML1 gene

A t(16;21)(q24;q22) translocation was detected by fluorescence in situ hybridization in a patient with acute myeloblastic leukemia previously treated for malignant lymphoma. While the breakpoint on chromosome 21 was within the AML1 gene as determined by FISH, the gene partner on chromosome 16 could not be identified. Band 16q24 appears to be rearranged in several types of myeloid proliferation and a review of the literature shows that these rearrangements most often occur in secondary leukemia and myelodysplastic syndrome or are part of complex chromosomal rearrangements.     

Application of cross-species color banding (RxFISH) in the study of T-prolymphocytic leukemia

BACKGROUND AND OBJECTIVE: Cross-species color banding (RxFISH) is a new FISH technology based on the use of differentially labeled gibbon chromosome probes to obtain a specific color banding pattern for each human chromosome. The aim of the study was to test the RxFISH technique for better characterization of complex karyotypes in patients with T-prolymphocytic leukemia (T-PLL). DESIGN AND METHODS: The study evaluated the cross-species color banding technique in four patients affected with T-PLL previously studied by conventional cytogenetics. RESULTS: All patients showed an abnormal karyotype and three of them had a complex karyotype. The involvement of 14q11 in all four cases, the gain of 8q in three cases and a loss of chromosome 10, 15 and 17 and a gain of chromosome 21 in two cases were noted. The RxFISH technique identified from 2 to 7 not previously recognized aberrations per case and confirmed the inv(14)(q11q32). INTERPRETATION AND CONCLUSIONS: To our knowledge, this is the first application of RxFISH to characterize chromosomal rearrangements in T-cell neoplasms. RxFISH gave rapid and easy identification of chromosome rearrangements that were difficult to recognize by conventional cytogenetics. Using this new technology we identified 15 rearrangements not detected by conventional cytogenetics.     

Comparison between conventional banding analysis and FISH screening with an AML-specific set of probes in 260 patients

Fluorescence in situ hybridization (FISH) is becoming popular in the diagnosis of clonal chromosomal abnormalities. We set up a fast FISH procedure using an extensive set of specific probes. Conventional banding analysis (CBA) and FISH were compared in 260 newly diagnosed acute myeloid leukemia (AML) patients. For FISH the following probes were used: MLL, CBF-beta/MYH11, ETV-6/AML1; AML1/ETO, BCR/ABL, PML/RAR, c-MYC, TP53, RB1, 5q31/5p15.2, 5q33-34, 7q31/CEP7, 20q13; CEP 4, X, Y. Result time was 96 h for CBA versus 5 h for FISH from direct harvest. CBA showed clonal abnormalities in 41% (n=105/260), normal karyotype in 39% (n=102/260) and failed in 20% (n=53/260). FISH screened all patients and detected abnormalities in 39% (n=102/260); CBA and FISH together identified abnormalities in 49% (n=128/260). In six patients with normal CBA and in eight patients with clonal karyotype, it detected further cryptic abnormalities. CBA showed clonal abnormalities in 13% of patients negative at FISH (n=21/158). FISH screening does not add relevant information to CBA, but is the quickest method for detecting major genetic abnormalities in AML. The speed of FISH is very valuable in AML-M3/M3v because PML/RAR+ patients require specific therapy. Furthermore, we suggest FISH screening in failed, complex or suboptimal quality chromosome and specific FISH analysis for 5q, 7q, 12p, 17p, inv(16), t(11q23) in order to implement CBA accuracy.     

New chromosome abnormalities and lack of BCL-6 gene rearrangements in Argentinean diffuse large B-cell lymphomas

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphomas. Cytogenetic studies have revealed a broad spectrum of clonal genetic abnormalities and complex karyotypes. The purpose of this study was to contribute to the understanding of the genomic alterations associated with this group of lymphomas. METHODS: Cytogenetic, fluorescence in situ hybridization (FISH) and molecular analyses were performed in 30 cases with DLBCL: 20 de novo DLBCL (dn-DLBCL) and 10 DLBCL secondary to follicular lymphoma (S-DLBCL). RESULTS: A total of 37 different structural chromosomal rearrangements were found: 27% translocations, 54% deletions, and 19% other alterations. Chromosomes 8, 6, 2, and 9 were the most commonly affected. Interestingly, translocation t(3;14)(q27;q32) and/or BCL-6 gene rearrangements were not observed either by cytogenetic studies or by FISH analysis. Fifteen novel cytogenetic alterations were detected, among them translocations t(2;21)(p11;q22) and t(8;18)(q24;p11.3) appeared as sole structural abnormalities. Translocation t(14;18)(q32;q21) and/or BCL-2-IGH gene rearrangements were the genomic alterations most frequently observed: 50% of S-DLBCL and 30% of dn-DLBCL. Deletions del(4)(q21), del(6)(q27), del(8)(q11), and del(9)(q11) were recurrent. The most common gains involved chromosome regions at 12q13-q24, 7q10-q32, and 17q22-qter; 6q was the most frequently deleted region, followed by losses at 2q35-qter, 7q32-qter, and 9q13-qter. Four novel regions of loss were identified: 5q13-q21, 2q35-qter (both recurrent in our series), 4p11-p12, and 17q11-q12. CONCLUSIONS: These studies emphasize the value of combining conventional cytogenetics with FISH and molecular studies to allow a more accurate definition of the genomic aberrations involved in DLBCL.     

Chromosome aberrations in a series of 120 multiple myeloma cases with abnormal karyotypes

We identified 120 multiple myeloma (MM) cases with satisfactory cytogenetic evaluation and abnormal karyotypes. Hyperdiploid karyotype was found in 77 cases (64%), hypodiploid in 30 cases (25%), and the remaining 13 cases (11%) had a pseudodiploid karyotype. The most common numerical abnormalities were gains of chromosomes 15, 9, 3 followed by chromosomes 19, 11, 7, 21, and 5. Whole chromosome losses were also frequent involving primarily chromosomes X/Y, 8, 13, 14, and 22. Most cases showed also structural rearrangements leading to del(1p), dup(1q), del(5q), del(6q), del(8p), del(9p), del(13q), and del(17p). Chromosome 13/13q deletion was found in 52% of cases; complete loss of 13 was observed in 73% of cases, whereas 27% had interstitial deletions. In addition, 13/13q deletions occurred in 75% of nonhyperdiploid myeloma but only 39% of the hyperdiploid had 13/13q deletions. Translocations affecting 14q32/IGH region was seen 40 cases; t(11;14)(q13;q32) in 17 cases, t(14;16)(q32;q23) and t(8;14)(q24;q32) in three cases each, and t(6;14)(p21;q32) and t(1;14)(q21;q32) in two cases each. The remaining 14q32 translocations had various t(V;14) partners or of an undetermined origin. Remarkably, the 14q32/IGH translocations were less frequent in the hyperdiploid karyotypes than the nonhyperdiploid karyotypes (17 vs. 63%). Fourteen cases showed break at 8q24/CMYC site; seven of those had Burkitt's-type translocations. Our results revealed that conventional cytogenetics remains an important tool in elucidating the complex and divers genetic anomalies of MM. Cytogenetics identifies two distinct groups of MM, hyperdiploid and nonhyperdiploid, and establishes the presence of prognostic chromosomal markers such as 13/13q, 17p, 8q24, and 16q aberrations.     

Cytogenetics of multiple myeloma: interpretation of fluorescence in situ hybridization results

The cytogenetic picture in multiple myeloma (MM) is highly complex, from which non-random numerical and structural chromosomal changes have been identified. Specifically, translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and either monosomy or deletions of chromosome 13 have been reported in a significant number of patients from both cytogenetic and interphase fluorescence in situ hybridization (FISH) studies. Importantly, these abnormalities of chromosome 13 have recently been associated with a poor prognosis. In view of the highly complex nature of the karyotypes in MM patients, interphase FISH results may be difficult to interpret. In this study, cytogenetics and/or interphase FISH were carried out on bone marrow samples or purified plasma cells from 37 MM patients. Abnormal karyotypes, characterized by multiplex FISH (M-FISH) were found in 11 patients, all of which were highly complex. Interphase FISH revealed translocations involving the IGH locus in 16 (43%) patients. The IGH/cyclin D1 (CCND1) gene fusion characteristic of the translocation, t(11;14)(q13;q32), was seen in 12 (32%) of these patients and other rearrangements of IGH in four (11%) patients. Fourteen patients had additional copies of chromosome 11. Twenty patients (54%) had 13q14 deletions, 10 of whom also had t(11;14) or another IGH translocation. By comparing cytogenetic and FISH results, this study has revealed that significant chromosomal abnormalities might be hidden within highly complex karyotypes. Therefore, extreme caution is required in the interpretation of interphase FISH results in MM, particularly in relation to certain abnormalities, such as 13q14 deletions, which have an impact on prognosis.     

Interphase fluorescence in situ hybridization and spectral karyotyping reveals hidden genetic aberrations in children with acute lymphoblastic leukaemia and a normal banded karyotype

Twenty-two cases of childhood acute lymphoblastic leukaemia (ALL) with normal G- or Q-banded karyotypes were studied by interphase fluorescence in situ hybridization (FISH) and spectral karyotyping. Probes detecting MLL, BCR/ABL and TEL/AML1 rearrangements were used for the interphase studies, along with centromere-specific probes from chromosomes 17 and X. In 10 patients (45%), previously undetected aberrations were demonstrable. Specific gene rearrangements and structural changes were found in six cases and numerical changes in five. Five of these aberrations have previously been reported to have an impact on prognosis. Three cases were massively hyperdiploid and, in one, the prognostically important BCR/ABL fusion was detected. In addition, a near-haploid karyotype with 27 chromosomes was found in one patient and TEL/AML1 rearrangements were detected in two cases. This study indicates that about half of childhood ALL cases with apparently normal karyotypes harbour genetic aberrations that may be detected using interphase FISH and spectral karyotyping.     

Fluorescence In Situ Hybridization Detection of Chromosome IGH/BCL2 Translocations from Paraffin-Embedded Tissue: Evaluation in Follicular Lymphoma

Follicular lymphoma is categorized as low-grade lymphoma because of the long median survival, but the disease is difficult to cure because of frequent recurrence. The t(14;18)(q32;q21) associated with follicular lymphoma juxtaposes a portion of BCL-2 (18q21) and IGH(14q32); the result is bcl-2 overexpression. In this study, a highly sensitive 2-color fluorescence in situ hybridization (FISH) method was used to detect t(14;18)(q32;q21) in the nuclei of paraffin-embedded tissue sections. Fourteen specimens, including 11 samples from follicular lymphoma and 3 samples from diffuse large cell lymphoma, for which results of karyotype study and paraffin-embedded tissues were available were selected for FISH study. The FISH results were compared with results of karyotype study of the lymph nodes involved in lymphoma. Among our 11 patients with the diagnosis of follicular lymphoma in whom karyotype study was performed, 8 had t(14;18)(q32;q21) in karyotype analysis, and 7 of these patients had a positive pattern in FISH analysis. In 1 case, FISH analysis was difficult because of weak signals. All 3 patients with diffuse large cell lymphoma and t(14;18)(q32;q21) in karyotype analysis had a positive pattern in FISH analysis. In 3 cases of follicular lymphoma without t(14;18)(q32;q21) in karyotype analysis, FISH did not show a positive pattern. Therefore the FISH assay in tissue was found to be very sensitive in detection of IGH/BCL2 translocation and was helpful in diagnosis of follicular lymphoma or in clarification of the cell origin of lymphoma when karyotype analysis was not available. Performing FISH on paraffin sections also is useful because we can identify cells with genetic abnormalities in the tumor and make a retrospective cytogenetic diagnosis even with old paraffin-embedded specimens. Int J Hematol. 2003;78:154-159.     

Development of a dual‐color,double fusion FISH assay to detect RPN1/EVI1 gene fusion associated with inv(3), t(3;3), and ins(3;3) in patients with myelodysplasia and acute myeloid leukemia

Approximately 2–3% of adult patients with acute myeloid leukemia harbor a rearrangement of RPN1 (at 3q21) and EVI1 (at 3q26.2) as inv(3)(q21q26.2), t(3;3)(q21;q26.2), or ins(3;3)(q26.2;q21q26.2). The most recent World Health Organization (WHO) classification has designated AML with inv(3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. We have created a dual color, double fusion fluorescence in situ hybridization (D‐FISH) assay to detect fusion of the RPN1 and EVI1 genes. A blinded investigation was performed using 30 normal bone marrow samples and 51 bone marrow samples from 17 patients with inv(3)(q21q26.2), 11 patients with t(3;3)(q21;q26.2), and one patient with ins(3;3)(q26.2;q21q26.2) previously defined by chromosome analysis. The unblinded results indicated abnormal RPN1/EVI1 fusion results in 30 (97%) of 31 samples from the inv(3)(q21q26.2) group including seven bone marrow samples for which chromosome analysis was unsuccessful or failed to detect an inv(3)(q21q26.2). Abnormal FISH results were detected in 14 (88%) of 16 samples with t(3;3)(q21;q26.2) and in the sole sample with an ins(3;3)(q26.2;q21q26.2). All 30 negative controls were normal and were used to establish a normal cutoff of 0.6% for the typical abnormal D‐FISH signal pattern. Overall, this D‐FISH assay was more accurate than chromosome analysis and based on the normal cutoff of 0.6%, this assay can be used for minimal residual disease detection and disease monitoring in patients with RPN1/EVI1 fusion. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.     

11例伴t(6;11)(q27;q23)急性白血病的临床和实验研究

目的　分析伴有t(6 ;11) (q2 7;q2 3)急性白血病 (AL)的形态学、免疫学、细胞遗传学和临床特点。方法　采用骨髓细胞直接法或短期培养法制备染色体 ,用R显带技术进行核型分析 ;采用双色混合谱系白血病 (MLL)基因探针和间期荧光原位杂交 (FISH)技术 ,对其中 10例AL进行MLL重排检测 ;分别用异硫氰酸荧光素 (FITC)和得克萨斯红 (Texasred)标记的 6号和 11号全染色体涂抹探针对其中 5例标本进行染色体研究。结果　t(6 ;11)易位病例主要见于急性髓系白血病(AML) M5(8/ 11例 )。 11例t(6 ;11)AL中 9例初诊时WBC计数 (10～ 10 0 )× 10 9/L之间 ,9例有不同程度的肝、脾、淋巴结浸润。 9例为单纯t(6 ;11) ,2例伴有其他异常。进行免疫表型分析的 9例白血病中 4例髓系和淋系抗原共表达 ,除 1例外 ,其余患者均有CD3 4 表达。本组t(6 ;11)患者中位生存期为 6个月。 10例患者的双色FISH研究显示均有MLL重排 ,其中 5例标本的涂抹分析也证实 6号和 11号染色体之间发生了相互易位。结论　t(6 ;11)AL有着独特的临床特点 ,其预后不良。染色体涂抹和间期双色FISH技术是检测该易位和MLL重排的可靠手段。     

The detection and significance of chromosomal abnormalities in childhood acute lymphoblastic leukaemia

In childhood acute lymphoblastic leukaemia (ALL), cytogenetics plays an essential role in diagnosis and prediction of outcome. Conventional cytogenetic analysis, complemented by fluorescence in situ hybridization (FISH), is highly effective in the accurate detection of chromosomal abnormalities. For the precise identification of specific genetic changes, molecular techniques may be applied. Chromosomal changes in ALL may be of structural or numerical type. A large number of established structural chromosomal rearrangements have now been described for which the genetic alterations and effect on prognosis are well known. These include t(9;22)(q34;q11) and BCR/ABL, rearrangements of 11q23 involving MLL, t(12;21)(p13;q22) with the ETV6/AML1 fusion, t(1;19)(q23;p13) with E2A/PBX1, t(8;14)(q24;q32) and the immunoglobulin genes. Genetic changes associated with T ALL are also known, although their effect on outcome is less pronounced. Rare chromosomal abnormalities are continually being discovered in small patient subgroups leading to the identification of new ALL associated genetic changes. Alterations in chromosome number have a strong impact on outcome in childhood ALL. The association of a high hyperdiploid karyotype (51-65 chromosomes) with a good prognosis has been known for more than 20 years. Conversely, the loss of chromosomes in the near-haploid group (23-28 chromosomes) indicates a poor outcome. New methods of cancer classification involving gene expression profiling may eventually supercede cytogenetic analysis in the diagnosis and prediction of outcome in leukaemia. It is more likely that they will be used in a complementary approach alongside cytogenetic, FISH and molecular analysis to guide patient management in childhood ALL.     

Comprehensive profile of cytogenetics in 2308 Chinese children and adults with de novo acute myeloid leukemia

Diagnostic cytogenetic and molecular analysis is recognized as the most valuable prognostic factor in acute myeloid leukemia (AML). Among 2516 consecutive Chinese patients with de novo AML, 2308 patients had successful cytogenetic results including 61 subclasses of cytogenetic abnormalities and 27 kinds of additional cytogenetic abnormalities. The incidence of t(15;17)(q22;q12) was highest (16.7% of 2308 patients), followed by t(8;21)(q22;q22) (15.1%), trisomy 8 (5.5%), loss of Y (4.5%), trisomy 21 (2.4%), inv(16)(p13q22) or t(16;16)(p13;q22) (2.1%), etc. In comparison to children, adults had higher incidence of normal karyotype (41.5% vs. 29.1%, P<0.001) and lower incidences of t(8;21)(q22;q22) (13.4% vs. 25.8%, P<0.001), t(9;11)(p22;q23) (0.2% vs. 1.2%, P=0.001) and other 11q23 rearrangements (1.0% vs. 3.4%, P<0.001). Among 349 AML patients with t(8;21)(q22;q22), 310 (35.5%) were found in 873 patients with M2. The t(15;17)(q22;q12) was exclusively observed in 386 (71.0%) of 544 patients with M3. In 48 AML patients with inv(16)(p13q22) or t(16;16)(p13;q22), 42 (15.2%) were detected in 276 patients with M4. Our study displayed the cytogenetic characteristics in a large series of Chinese patients with de novo AML. Our results revealed the similarities and differences of cytogenetic abnormalities existing between Chinese and western AML patients.