Four novel non-random chromosome rearrangements in B-cell chronic lymphocytic leukaemia: 6p24-25 and 12p12-13 translocations, 4q21 anomalies and monosomy 21

Nine patients with previously unreported chromosome changes were identified among 209 B-cell chronic lymphocytic leukaemia (CLL) cases: three patients had a translocation involving 6p24-25; three had a 12p12-13 translocation; two had 4q21 involvement (one with coexisting 6p anomaly); and two had monosomy 21. Interphase fluorescence in situ hybridization (FISH) detected some cryptic aberrations (+12, 6q-, 17p-, 11q-) in those patients with 6p translocations, whereas only a cytogenetically undetected 13q14 deletion was found in the remaining cases. Atypical morphology was noted in six cases, including both cases with monosomy 21, two cases with 6p and 4q21 anomaly and one case with 12p involvement. Four of these cases also had more than one phenotype deviation with respect to the classical CLL phenotype. Disease progression after 21-51 months (median 41) was noted in two cases with 6p and 4q21 involvement and in one case with 12p anomaly and monosomy 21. We arrived at the following conclusions: (i) 6p24-25 and, possibly, 4q21 lesions represent non-random events in CLL, occurring in association with other well-known unbalanced rearrangements; (ii) 12p rearrangements and monosomy 21 may possibly represent early chromosome defects that are not associated with the classical DNA gains and losses known to be present in the majority of CLL; and (iii) atypical morphology and immunophenotype as well as disease progression were frequently observed in these cases     

Structural aberrations of chromosomes 17 and 12 in chronic B-cell disorders

OBJECTIVES: Genomic aberrations can now be identified in approximately 80% of chronic lymphocytic leukemia, small lymphocytic lymphoma (CLL/SLL) patients. In the present study, four new structural changes involving chromosomes 17 and 12 in CLL/SLL patients are described. METHODS: Five patients were selected for inclusion in the present report among a total of 92 cases with diagnosis of CLL/SLL. Cytogenetic studies and fluorescence in situ hybridization (FISH) analysis to detect some of the most frequent cryptic aberrations occurring in CLL/SLL patients were performed. Clinical studies are also described. RESULTS: Four cases showed structural rearrangements of chromosome 17. A psu dic(17;2)(p11.2;p21), leading to p53 deletion, was observed in a patient who developed a mixed cellularity Hodgkin's disease coexisting with the CLL/SLL in the same lymph node. Epstein Barr virus was detected in the Reed-Sternberg cells. Two cases had a balanced translocation t(2;17)(p21;q23). Both patients showed trisomy 12 and clonal evolution and one of them also had 11q deletion. In addition, a der(17)t(12;17)(q13;q25) as a part of a complex karyotype, and a complex translocation t(5;12;19) (q15;p11;q13) were also found. Four patients had an adverse clinical outcome and died because of disease progression. CONCLUSIONS: Four unreported nonrandom chromosome aberrations in CLL/SLL patients, one of them who might represent a new recurrent abnormality, are described. These uncommon abnormalities, mostly associated with evolving disease, may have implications for the understanding of genetic events associated with disease progression in this pathology.     

The t(14;19)(q32;q13)-positive small B-cell leukaemia: a clinicopathologic and cytogenetic study of seven cases

The t(14;19)(q32;q13), involving the BCL3 locus at chromosome 19q13 and the immunoglobulin heavy chain gene at 14q32, is a rare recurrent cytogenetic abnormality identified in B-cell neoplasms, most of which have been classified as chronic lymphocytic leukaemia (CLL) in the literature. We describe the clinicopathological, immunophenotypic and cytogenetic findings in seven patients with B-cell neoplasms associated with t(14;19)(q32;q13). There were five men and two women, with a median age of 48 years (range 33-68). All had absolute lymphocytosis, six had lymphadenopathy, and one had splenomegaly. Lymphocytes in blood and bone marrow aspirate smears were predominantly small and cytologically atypical. Flow cytometric immunophenotyping showed an atypical immunophenotype with low CLL scores. The growth pattern in bone marrow biopsy specimens was interstitial to diffuse; immunohistochemical stains were positive for bcl3 and negative for cyclin D1. Lymph node biopsy specimens of two patients revealed total architectural effacement by neoplasm with proliferation centres. In addition to t(14;19), cytogenetic studies demonstrated trisomy 12 in five patients. These results suggest that B-cell neoplasms with the t(14;19)(q32;q13) present frequently as leukaemia composed of small B-lymphocytes and share many features with CLL. However, these neoplasms also differ from CLL cytologically and in their immunophenotype.     

Cyclin D3 at 6p21 is dysregulated by recurrent chromosomal translocations to immunoglobulin loci in multiple myeloma.

Reciprocal chromosomal translocations, which are mediated by errors in immunoglobulin heavy chain (IgH) switch recombination or somatic hypermutation as plasma cells are generated in germinal centers, are present in most multiple myeloma (MM) tumors. These translocations dysregulate an oncogene that is repositioned in proximity to a strong IgH enhancer. There is a promiscuous array of nonrandom chromosomal partners (and oncogenes), with the 3 most frequent partners (11q13 [cyclin D1]; 4p16 [FGFR3 and MMSET]; 16q23 [c-maf]) involved in nearly half of MM tumors. It is now shown that a novel t(6;14)(p21;q32) translocation is present in 1 of 30 MM cell lines and that this cell line uniquely overexpresses cyclin D3. The cloned breakpoint juxtaposes gamma 4 switch sequences with 6p21 sequences that are located about 65 kb centromeric to the cyclin D3 gene. By metaphase chromosome analysis, the t(6;14) (p21;q32) translocation was identified in 6 of 150 (4%) primary MM tumors. Overexpression of cyclin D3 messenger RNA (mRNA) was identified by microarray RNA expression analysis in 3 of 53 additional primary MM tumors, each of which was found to have a t(6;14) translocation breakpoint by interphase fluorescence in situ hybridization analysis. One tumor has a t(6;22)(p21;q11) translocation, so that cyclin D3 is bracketed by the IgL and IgH breakpoints. These results provide the first clear evidence for primary dysregulation of cyclin D3 during tumorigenesis. It is suggested that the initial oncogenic event for most MM tumors is a primary immunoglobulin translocation that dysregulates cyclin D1, cyclin D3, and other oncogenes to provide a proliferative stimulus to postgerminal center plasma cells.     

Loss of heterozygosity on chromosome 10q22-10q23 and 22q11.2-22q12.1 and p53 gene in primary hepatocellular carcinoma

AIM: To analyze loss of heterozygosity (LOH) and homozygous deletion on p53 gene (exon2-3, 4 and 11), chromosome 10q22-10q23 and 22q11.2 -22q12.1 in human hepatocellular carcinoma (HCC). METHODS: PCR and PCR-based microsatellite polymorphism analysis techniques were used. RESULTS: LOH was observed at D10S579 (10q22-10q23) in 4 of 20 tumors (20%), at D22S421 (22q11.2-22q12.1) in 3 of 20(15%), at TP53.A (p53 gene exon 2-3) in 4 of 20(20%), at TP53.B (p53 gene exon 4) in 6 of 20(30%), and at TP53.G (p53 gene exon 11) in 0 of 20(0%). Homozygous deletion was detected at 10q22-10q23(8/20; 40%), 22q11.2-22q12.1(8/20; 40%), p53 gene exon 2-3(0/20;0%), p53 gene exon 4(6/20; 30%), and p53 gene exon 11(2/20; 10%). CONCLUSION: There might be unidentified tumor suppressor genes on chromosome 10q22-10q23 and 22q11.2-22q12.1 that contribute to the pathogenesis and development of HCC.     

Initial clonal acute lymphoblastic transformation of chronic lymphocytic leukemia with (11;14) and (8;12) chromosome translocations and acquired homozygosity

A 69-year-old woman presented with chronic lymphocytic leukemia (CLL) diagnosed after bone marrow recovery following intensive chemotherapy for acute lymphoblastic leukemia. The retrospective diagnosis of initial blastic transformation of CLL was made. The patient eventually died, 5 months after initial blastic phase, from a blastic relapse of her CLL. Immunologic and karyotypic studies of the lymphoid cells at different times during the evolution of the disease provided evidence that both the small lymphocytes and the large blastic cells originated from the same clone since they carried the same surface markers and the same (11;14) (q13;q32) translocation. A subpopulation of cells carried in addition a t(8;12) (q24;q22) together with the loss of the normal chromosome 8 and homozygosity for the abnormal der(8). The possibility that this second population might correspond to the blast cells and that the occurrence of the t(8;12) with homozygosity might be linked to blastic transformation of the CLL is discussed.     

Dic(17;18)(p11.2;p11.2) is a recurring abnormality in chronic lymphocytic leukaemia associated with aggressive disease

Interphase cytogenetics are commonly used to identify clonal abnormalities in chronic lymphocytic leukemia (CLL) patients but fail to identify recurrent translocations that ultimately can direct more focused molecular characterization. Given the importance of del(17p13.1) in CLL outcome, we performed an extensive review of 1213 patients undergoing metaphase cytogenetics at our institution and identified 16 (1·3%) with a recurrent unbalanced translocation between the p arms of chromosomes 17 and 18 that results in a dicentric chromosome with loss of much of 17p and 18p. The dic(17;18)(p11.2;p11.2) was associated with a complex (three or more unrelated cytogenetic abnormalities) karyotype in 12 patients (75%) at the time that the abnormality was first identified, and eventually associated with a complex karyotype in 94% of patients. IGHV mutational analysis was un‐mutated in 88% of cases where evaluation was possible. Except for one patient who was diagnosed with CLL incidentally during a workup for metastatic tonsillar cancer, all patients identified with dic(17;18)(p11.2;p11.2) met criteria for disease treatment, with a median time from diagnosis to first treatment of 15 months. Our data demonstrate that dic(17;18)(p11.2;p11.2) is a novel recurrent cytogenetic abnormality in CLL associated with early age at diagnosis and accelerated disease progression. Future efforts to identify genes disrupted by this translocation are warranted and ongoing.     

The chromosome translocation (11;14)(p13;q11) associated with T-cell acute lymphocytic leukemia: an 11p13 breakpoint cluster region

The translocation (11;14)(p13;q11) was observed in karyotypes of leukemic cells from a 3-year-old boy with T-cell acute lymphocytic leukemia (T-ALL). Since this translocation is a recurrent marker of T-ALL, we undertook to investigate its mode of formation and role in leukemogenesis. The cytogenetic breakpoint on chromosome 14 occurs in 14q11, the same band wherein lies the T-cell receptor alpha/delta chain gene; and Southern hybridization analysis of peripheral blood and bone marrow DNA uncovered a tumor-specific rearrangement in the D delta-J delta region of this locus. DNA encompassing the rearrangement was isolated by molecular cloning, and further analysis revealed it to be the t(11;14)(p13;q11) junction. Nucleotide sequence determination of the junction indicates that the 14q11 breakpoint occurs immediately adjacent to the D delta 2 gene segment. Hence, the translocation arose as an aberrant rearrangement between the downstream recombination signal of D delta 2 and a pseudo recombination signal adjacent to the chromosome 11 breakpoint. Finally, comparison of the breakpoint in band 11p13 with those of other translocations (11;14)(p13;q11) identified a breakpoint cluster region of approximately 1.2 kilobase-pairs (kb), alterations of which may promote the development of T-ALL.     

Association and clonal distribution of trisomy 12 and 13q14 deletions in chronic lymphocytic leukaemia

The coexistence of trisomy 12 and deletions of chromosome 13 (13q12-q32) has rarely been observed in chronic lymphocytic leukaemia (CLL). Fluorescence in situ hybridization (FISH) performed on 600 consecutive CLL patients revealed the association of trisomy 12 and 13q14 deletion, of at least one of the three markers analysed (RB1, D13S319 and D13S25), in 55 cases (9% of 600 and 46% of 120 trisomy 12 cases). Trisomy 12 and isolated RB1 deletion were seen in 14/120 cases, trisomy 12 and D13S319/D13S25 deletion with diploid RB1 in 19/118, and trisomy 12 and deletion encompassing the three 13q markers studied in 22/118 cases. The heterogenous distribution of trisomy 12 and 13q deletions within the neoplastic B cells suggests that they are secondary rather than primary events in CLL leukaemogenesis.     

G-banding and molecular cytogenetic analyses of marginal zone lymphoma

We analysed the acquired chromosomal aberrations of 22 marginal zone lymphoma (MZL) patients by various genome-wide cytogenetic techniques, such as G-banding, multicolour fluorescence in situ hybridisation (M-FISH), cross-species colour banding (RxFISH), and comparative genomic hybridisation (CGH), as well as FISH with locus-specific probes. Patients with an abnormal chromosome 3 (n = 11), the most frequently rearranged chromosome, showed a shorter median survival than patients with a normal chromosome 3 (n = 11, 74 months vs. 219 months, P < 0.03). Four of five patients with nodal MZL had chromosome 3 abnormalities and patients with nodal MZL had a shorter median survival than patients in the other morphological subgroups of MZL (P < 0.003). CGH analysis showed only gains of chromosome material, namely of chromosome regions 3p12-25, 3q12-21, 3q23-28, 12q13-15, 12q22-24, 19p13 and 19q13 in two to four cases each (20-40%). In two MZL, the novel unbalanced translocation der(13)t(3;13)(q24;p11) was detected as the sole karyotypic rearrangement, indicating that gain of 3q24-qter could be an important event in the pathogenesis of these lymphomas. Another two cases showed, in addition to other abnormalities, a t(4;14)(p13;q32). Both these lymphomas had involvement of the IGH gene at 14q32, and one of them also of the RHOH/TTF gene at 4p13, which encodes a new member of the RHO protein subfamily.     

Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences

Cyclin D1 expression is deregulated by chromosome translocation in mantle cell lymphoma and a subset of multiple myeloma. The molecular mechanisms involved in long-distance gene deregulation remain obscure, although changes in acetylated histones and methylated CpG dinucleotides may be important. The patterns of DNA methylation and histone acetylation were determined at the cyclin D1 locus on chromosome 11q13 in B-cell malignancies. The cyclin D1 promoter was hypomethylated and hyperacetylated in expressing cell lines and patient samples, and methylated and hypoacetylated in nonexpressing cell lines. Domains of hyperacetylated histones and hypomethylated DNA extended over 120 kb upstream of the cyclin D1 gene. Interestingly, hypomethylated DNA and hyperacetylated histones were also located at the cyclin D1 promoter but not the upstream major translocation cluster region in cyclin D1-nonexpressing, nontumorigenic B and T cells. RNA polymerase II binding was demonstrated both at the cyclin D1 promoter and 3' immunoglobulin heavy-chain regulatory regions only in malignant B-cell lines with deregulated cyclin D1 expression. Our results suggest a model where RNA polymerase II bound at IgH regulatory sequences can activate the cyclin D1 promoter by either long-range polymerase transfer or tracking.     

Concurrent activation of a novel putative transforming gene, myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32)

Through the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization. Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5), either the 5' E(mu) enhancer or the most telomeric 3' Ealpha enhancer was juxtaposed to myeov. Although cyclin D1 overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated. (Blood. 2000;95:2691-2698)     

Amplification of multiple regions of chromosome 12, including 12q13–15, in chronic lymphocytic leukaemia

Abstract: Trisomy 12 is a frequent abnormality in chronic lymphocytic leukaemia (CLL). The biological importance of trisomy 12 is still poorly understood but it has been suggested that one or several genes are duplicated leading to malignant transformation. We present a case with amplification of 12q13–22 found in a clinically aggressive relapse of CLL. A smaller region, 12q13–15, was amplified most frequently and a YAC containing the MDM2 gene gave the highest number of signals. Additionally, in a subclone an amplicon containing at least 5 copies of a cosmid from 12q23–24 was detected. The case shows that small duplications of chromosome 12, not revealed by cytogenetic analysis, may occur in CLL. Also, it shows that cytogenetic clonal evolution can occur in CLL without morphological evidence of blast transformation. Our results indicate that the 12q13–15 region carries an important gene for CLL progression.     

TEL gene is involved in myelodysplastic syndromes with either the typical t(5;12)(q33;p13) translocation or its variant t(10;12)(q24;p13)

A t(5;12)(q33;p13) translocation is a recurrent chromosome abnormality in a subgroup of myeloid malignancies with features of both myeloproliferative disorders and myelodysplastic syndromes (MDSs). The molecular consequence of a t(5;12) is a fusion between the platelet- derived growth factor receptor-B gene on chromosome 5 and a novel ETS- like gene, TEL, on chromosome 12. We report on three patients with a t(5;12)(q33;p13) diagnosed as chronic myelomonocytic leukemia, and one case of a t(10;12)(q24;p13) in a progressive MDS, with eosinophilia and monocytosis. Involvement of the TEL gene in these chromosome translocations was investigated by fluorescence in situ hybridization (FISH) with cosmid probes containing selectively the 5' end or 3' end of TEL. Hybridization of these cosmids to the der(5)/der(10) or a der(12), respectively, demonstrated a rearrangement of TEL in both translocations, showing that the t(10;12) is a variant translocation of the t(5;12). Cloning of the fusion cDNA of one case of t(5;12) showed that the breakpoint occurred at the RNA level at exactly the same position as reported by Golub et al (Cell 77:307, 1994). In addition, the TEL gene on chromosome 12 could be localized between two probes previously mapped to 12p13, namely PRB1 and D12S178, leading to a better definition of the position of TEL in this chromosome region. Moreover, in the case involving chromosome 10, the breakpoint occurred between cKTN206 and cKTN312/LYT-10 at 10q24. Clinicohematological data in these studies as well as the restriction mapping of chromosomal breakpoints strongly suggest that (1) common features in MDSs involving the TEL gene are monocytosis and eosinophilia, (2) chromosomes other than no. 5 may be involved and at least a t(10;12)(q24;p13) variant chromosome translocation does exist in these MDSs, and (3) both standard and variant 12p/TEL translocations may be identified by FISH with appropriate probes.     

Novel recurrent genetic imbalances in human hepatocellular carcinoma cell lines identified by comparative genomic hybridization

To search for recurrent and specific genomic alterations in human hepatocellular carcinoma (HCC), we examined 18 cell lines by comparative genomic hybridization (CGH), a molecular cytogenetic approach that allows positional identification of gains and losses of DNA sequences of the entire tumor genome. We report here a distinct pattern of multiple recurrent DNA copy-number gains and losses that include alterations frequently seen in other neoplasias as well as changes potentially specific for HCC. The most frequent gains were localized on 1p34.3-35, 1p33-34.1, 1q21-23, 1q31-32, 6p11-12, 7p21, 7q11.2, 8q24.1-24.2, 11q11-13, 12q11-13, 12q23, 17q11. 2-21, 17q23-24, and 20p11.1-q13.2. Recurrent losses were mapped on 3p12-14, 3q25, 4p12-14, 4q13-34, 5q21, 6q25-26, 8p11.2-23, 9p12-24, 11q23-24, 13q12-33, 14q12-13, 15q25-26, 18q11.2-22.2, and 21q21-22. Seventeen genomic imbalances are novel in HCC, thus extending significantly the map of genetic changes and providing a starting point for the isolation of new genes relevant in pathogenesis of liver neoplasia, as well as providing molecular probes for both diagnosis and monitoring treatment of the disease.     

An additional breakpoint region in the BCL-1 locus associated with the t(11;14)(q13;q32) translocation of B-lymphocytic malignancy

The t(11;14)(q13;q32) translocation is associated with human B- lymphocytic malignancy. This translocation divides the IgH locus on chromosome 14q32 and may activate a postulated proto-oncogene, bcl-1, located on chromosome 11q13. Two samples of chronic lymphocytic leukemia with the t(11;14)(q32;q13) translocation were studied. The break in one sample was shown to join Jh sequences with the previously described bcl-1 major translocation cluster. DNA blots of the second sample suggested that Jh sequences were joined to a different breakpoint region on chromosome 11. This translocation was cloned and found to link the human Jh3 region and a new breakpoint region 63 kb telomeric of the major translocation cluster. This translocation occurred in part as the result of an aberrant D-J recombination. Recurrent translocations human B-lymphocytic malignancy. The definition of a new breakpoint region may aid the identification of the postulated bcl-1 gene.