t(14;22)(q32;q11) in non-Hodgkin lymphoma and myeloid leukaemia: molecular cytogenetic investigations

Two non-Hodgkin lymphomas (NHL), one chronic lymphocytic leukaemia/small lymphocytic lymphoma and one diffuse large B-cell lymphoma and three cases of myeloid leukaemia, two chronic (CML) and one acute (AML), showed, by G-banding analysis, apparently identical chromosomal translocations t(14;22)(q32;q11), in three of the cases as the sole abnormality. Fluorescence in situ hybridisation (FISH) analysis with locus-specific probes for ABL at 9q34 [bacterial artificial chromosomes (BACs) 835J22 and 1132H12], IGH at 14q32 [P1 artificial chromosome (PAC) 998D24] and IGL (PAC 1019H10) and BCR (BAC 74M14) at 22q11, as well as multicolour in situ hybridisation (M-FISH) analyses were performed. A three-way variant translocation of the classical t(9;22)(q34;q11), t(9;22;14)(q34;q11;q32), involving both BCR and ABL, was unravelled by the molecular cytogenetic investigations in the three myeloid leukaemia cases; a similar variant translocation has previously been reported in seven CML. The two cases of NHL (one NHL with a similar 14;22-translocation has been reported previously) had no involvement of BCR or ABL, but instead the IGH and IGL genes were shown to be juxtaposed by the t(14;22)(q32;q11). How such a rearrangement with recombination of IGH and IGL might elicit a pathogenetic effect is completely unknown.     

An additional segment at 1p36 derived from der(18)t(14;18) in patients with diffuse large B-cell lymphomas transformed from follicular lymphoma

The particular translocation in follicular lymphomas (FLs) is a t(14;18)(q32;q21), recombining the immunoglobulin heavy chain (IgH) gene on chromosome 14 with the B-cell leukemia/lymphoma 2 (BCL2) gene on chromosome 18. Some low-grade FLs are aggressively transformed into diffuse large B-cell lymphomas, presumably by acquisition of secondary chromosomal changes, including chromosomal band 1p36. A common example is add(1)(p36). Because it is difficult to identify the origin of add(1)(p36) even on high-resolution G-banding analysis, we used spectral karyotyping (SKY) and double-color fluorescence in situ hybridization (DC-FISH) to define the t(14;18) and the extra band at 1p36 in two cases of diffuse large B-cell lymphoma (DLBCL). SKY revealed that the extra chromosomal segment on 1p36 was derived from chromosome 18. DC-FISH defined BCL2/IgH fusion signals at 1p36 in addition to t(14;18), suggesting that BCL2/IgH fusion at 1p36 was an evolutionary alteration following the primary BCL2/IgH translocation on der(18) in both cases. Our results indicate that IgH alleles, implicated in chromosomal rearrangement, may themselves frequently be targets for secondary translocations, suggesting that multiple IgH translocations and insertions are associated with the progression of FL.     

Four new recurring translocations in non-Hodgkin lymphoma

The identification of recurring chromosomal translocations has provided clues to the gene regions important in lymphoma development. Among 157 patients with non-Hodgkin lymphoma studied by cytogenetic analysis, four new recurring translocations have been identified--t(8;9) (q24;p13), t(11;18)(q21;q21), t(14,15)(q32;q15), and an unbalanced translocation giving rise to der(22)t(17;22) (q11;p11). Each translocation appeared twice. The t(11;18) was the only karyotypic abnormality in the two patients with it, and the t(14;15) was the sole karyotypic abnormality in one patient. All translocations were found in B-cell malignancies and were associated with both nodal and extranodal disease. Among the regions affected, only the immunoglobulin heavy- chain gene MYC, and BCL2, have thus far been associated with lymphoma. The breakpoint sites identified by these translocations warrant further investigation at the molecular level.     

Translocations of 14q32 and deletions of 13q14 are common chromosomal abnormalities in systemic amyloidosis

Systemic monoclonal immunoglobulin light chain amyloidosis (AL) is associated with clonal plasma cell dyscrasias that are often subtle and non-proliferating. AL shares numerical chromosomal changes with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Illegitimate translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and deletions of the long arm of chromosome 13, [del(13q)], commonly occur in MM, MGUS and plasma cell leukaemia. In AL IGH rearrangements have been identified but, to date, there are no reports of del(13q). In this study of 32 patients with AL, 24 with systemic and eight with localized disease, translocations involving IGH and del(13q) were found using dual-colour interphase fluorescence in situ hybridization (FISH). IGH translocations were observed in 11 patients (37% overall and in 46% with systemic disease), of which nine had the IGH/CCND1 fusion from t(11;14)(q13;q32). Two showed IGH translocations other than the t(11;14) or t(4;14)(p16;q32). In one of these patients a breakpoint within the constant region of IGH between Calpha1 and Calpha2 was indicated. In the second a deletion covering Calpha1 and Calpha2 accompanied the translocation. Ten patients (27% overall and 33% of those with systemic disease) showed del(13q). The gain or loss of IGH and CCND1 signals provided evidence of numerical chromosomal changes in three patients.     

Cytogenetic and molecular delineation of a region of chromosome 3q commonly gained in marginal zone B-cell lymphoma

BACKGROUND AND OBJECTIVES: Whole or partial trisomy 3 represents the most recurrent chromosomal abnormality occurring in marginal zone B-cell lymphoma (MZBCL), a distinct subtype of B-cell non-Hodgkin's lymphoma (NHL). By conventional cytogenetic analysis, unbalanced translocations involving chromosome 3 and leading to a partial trisomy 3q were identified in a series of 14 MZBCL patients. Fluorescent in situ hybridization (FISH) experiments were then performed to characterize the breakpoints further and to delineate the extent of the 3q gained region more accurately. DESIGN AND METHODS: We studied 14 cases of MZBCL combining cytogenetics and FISH techniques using specific probes for the long arm of chromosome 3, including the chromosome 3 a satellite probe, a representative panel of yeast artificial chromosome (YAC) clones mapping the chromosomal 3q region (3q11.2 to 3q23) and the chromosome 3 subtelomeric (3q29) probe. RESULTS: In the 14 cases, additional chromosome 3q material was found to be involved in different unbalanced translocations with chromosomes 1, 6, 7, 8, 11, 13, 14, 15, 17, 19 and 21, leading to a derivative chromosome. None of the chromosomal abnormality juxtaposed the 3q regions with the heavy and/or light k and l immunoglobulin gene loci. Eight different breakpoints distributed between the 3q11.2 and the 3q13.32 regions were identified and a common 3q13.32 3q29 overrepresented region was delineated. INTERPRETATION AND CONCLUSIONS: These results suggest that this critical region may be of importance in the pathogenesis of MZBCL and support the hypothesis that a gene dosage effect rather than a specific gene disruption may be involved in the development of this disease.     

Biallelic DNA rearrangements and deletions within the BCL-6 gene in B-cell non-Hodgkin's lymphoma

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B-cell neoplasms, and the BCL-6 gene, identified on 3q27, was shown to be disrupted and over-expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL-6 gene in seven out of 3 5 cases with B-cell non-Hodgkin's lymphoma (NHL). Further analysis revealed that three of these patients with BCL-6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL-6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL-6 abnormality.     

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies.

Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-kappaB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms of BCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues. BCL11A was also highly homologous to another gene (BCL11B) on chromosome 14q32.1. BCL11A coamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting that BCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.     

Chromosome translocations involving band 7q35 or 7p15 in childhood T- cell leukemia/lymphoma

In a chromosome study in childhood T-cell leukemia/lymphoma, we found t(7;11)(q35;p13) in 2 patients, t(7;14) (q35;q11) in one patient, and t(7;14)(p15;q32) in 1 patient. Southern blotting and in situ chromosomal hybridization studies in one patient with the t(7;11) demonstrated that both alleles of the T-cell antigen receptor beta- subunit gene (TCRB) were rearranged, and that one TCRB allele had relocated from 7q35 to the fusion point in band p13 of the involved chromosome 11 (11p-). These findings suggest that juxtaposition of TCRB with the putative oncogene tcl-2 located in band 11p13 may be a critical step toward development of this T-cell leukemia/lymphoma. In the other two translocations, all breakpoints were sites for lymphocyte function genes, ie, 7q35 for TCRB, 14q11 for T-cell antigen receptor alpha-subunit gene (TCRA), 7p15 for T-cell antigen receptor alpha- subunit gene (TCRG), and 14q32 for immunoglobulin heavy-chain gene (IGH). Thus, the findings in these cases allow us to expand the above hypothesis and propose that the juxtaposition of TCRB or TCRG with tcl- 2, TCRA, or IGH through chromosomal translocation may activate a mechanism for the genesis of T-cell leukemia/lymphoma with these chromosome translocations.     

Identification of the gene encoding cyclin E1 (CCNE1) as a novel IGH translocation partner in t(14;19)(q32;q12) in diffuse large B-cell lymphoma

In a subset of B-cell malignancies, the genes encoding members of the cyclin D familiy are juxtaposed to immunoglobulin loci through recurrent chromosomal translocations. Here, we identified the gene encoding cyclin E1 as novel translocation partner of the immunoglobulin heavy chain (IGH) locus involved in a t(14;19)(q32;q12) in a case of t(8;14)(q24;q32) IGH-MYC-positive leukemic diffuse large B-cell lymphoma. The translocation breakpoints were cloned and mapped to the switch region Sα1 of IGH in 14q32 and approximately 60kb centromeric to CCNE1 in 19q12. Immunohistochemical analysis revealed overexpression of the cyclin E1 protein in this case, which to a comparable extent was observed in 3/41 independent DLBCL. These data indicate that cyclin E1 may act as a novel oncogene in B-cell lymphomagenesis.     

Detection of 14q32 translocations in B-cell malignancies by in situ hybridization with yeast artificial chromosome clones containing the human IgH gene locus

Partner sites of 14q32 translocations found in B-cell malignancies were detected by fluorescence in situ hybridization (FISH) using yeast artificial chromosome (YAC) clones, Y20 and Y6, containing the human Ig heavy chain (IgH) gene locus. Y20 spans a 160-kb upstream and 40-kb downstream region of the JH segments on chromosome band 14q32.33. Y6 is 300-kb upstream of Y20, and spans a further 320-kb telomeric region. The human DNA sequences amplified by Alu polymerase chain reaction of the YAC clones were used as probes for FISH to study six patients with non-Hodgkin's lymphoma (NHL), one patient with acute lymphoblastic leukemia, and one cell line FR4 established from a plasmacytoma. Three telomeric YAC clones each specific for 3q, 8q, and 18q were also used to further characterize 14q32 translocations. The IgH YACs were successfully applied to detect cytogenetically invisible subtelomeric translocation of the IgH gene locus to each partner site in t(14;18), t(8;14), and t(14;19), and to identify t(3;14) (q27;q32.33) in three patients with 14q32 translocation of unknown origin. Furthermore, complex translocations involving more than three chromosomes were detected in an NHL patient with t(8;14), and t(3;12), and in the FR4 with der(14)t(8;14), der(8)dic(1;8), and del(1)(q21). The technique would be a useful tool in elucidating the mechanisms of a 14q32 translocation in B-cell malignancies.     

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.     

Translocations involving band 3q27 and Ig gene regions in non-Hodgkin's lymphoma.

We report a series of 20 non-Hodgkin's lymphomas (NHL) in which cytogenetic analysis showed a translocation involving band 3q27 and the site of one of the three Ig genes (14q32, 2p12, 22q11) in the neoplastic cells. These cases were found in a series of 319 patients with clonal chromosomal abnormalities studied over a 7-year period. Fourteen patients had diffuse lymphoma, mainly of large cell type and the remaining six were follicular lymphomas. All cases studied were of B-cell phenotype. A t(3;14)(q27;q32) was commonest, found in 15 patients (4.7%), with the two variant translocations, t(3;22)(q27;q11) and t(2;3)(p12;q27), being found in three and two patients, respectively. Additional chromosomal defects were present in most patients, but two patients had this type of translocation as the sole abnormality. These results indicate that translocations involving band 3q27 and Ig genes are not uncommon, and suggest that a novel oncogene, located at band 3q27, may be implicated in B-cell NHL.     

Rearrangement of the chromosome 11 bcl-1 locus in centrocytic lymphoma: analysis with multiple breakpoint probes

Centrocytic lymphoma is a B-cell non-Hodgkin's lymphoma (NHL) composed of lymphocytes resembling cleaved follicular center cells (centrocytes). Previous studies have suggested an association between t(11;14) chromosomal translocations and bcl-1 rearrangement in centrocytic and related intermediate lymphocytic lymphomas. To further characterize the association between bcl-1 and centrocytic lymphoma, Southern blot analysis was performed on samples from 23 patients using four separate bcl-1 breakpoint probes spanning 63 kb of the chromosome 11 bcl-1 locus. Rearrangements were identified in six patients with the major translocation cluster (MTC) probe and in another six with probe p94PS, located about 24 kb 5' of MTC. Eleven of these 12 cases showed comigration of rearranged bcl-1 and Ig heavy chain-joining genes, consistent with the t(11;14) chromosomal translocation. No rearrangements were observed with the bcl-1 locus probes p210 or p11EH located 5' of p94PS, nor with bcl-2 or c-myc oncogene probes. No bcl-1 rearrangements were identified in B-cell follicular NHL (15), small noncleaved cell (Burkitt's and non-Burkitt's) NHL (8), T-cell NHL (4), multiple myeloma (14), and pre-B-cell acute lymphoblastic leukemia (9). One of 23 B-cell NHL of large cell type and one of 19 chronic lymphocytic leukemias or small lymphocytic NHL had MTC rearrangement. Thus, bcl-1 rearrangement occurred at MTC or p94PS in 12 of 23 centrocytic lymphomas (52%), confirming a nonrandom association and suggesting a pathogenetic role for the bcl-1 locus in this immunohistologic subtype of NHL.     

Localization of human immunoglobulin kappa light chain variable region genes to the short arm of chromosome 2 by in situ hybridization.

The genes for human immunoglobulin kappa light chains have been localized in normal lymphocyte and fibroblast chromosomes by in situ hybridization of probes from cloned DNA fragments of the kappa variable region locus. The localization was achieved by counting grains (after autoradiography) over chromosomes in a number of karyotypes. The variable region gene probes hybridized in a cluster on a region of the chromosome 2 short arm close to the centromere (2cen leads to p12). This location was confirmed in lymphocytes from a balanced translocation carrier 46XXt (2; 16) (q13; q22). Our results show that human kappa light chain genes are located in the region of the break point observed in specific chromosomal translocations associated with Burkitt lymphoma.     

Interphase and metaphase detection of the breakpoint of 14q32 translocations in B-cell malignancies by double-color fluorescence in situ hybridization

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24-68, containing VH segments. CY24-68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24-68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24-68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.