BCL-6 and other genomic alterations in non-Hodgkin's lymphoma (NHL).

This study reports on the frequency and disease association pattern of a number of gene rearrangements in a large panel of lymphoid tumours (n = 94). We detected the t(11;14) translocation, involving rearrangement of the BCL-1 locus, in 60% of mantle cell lymphomas. The BCL-2 gene, located at band 18q21, was rearranged in 42% of follicle centre lymphomas (FCL) and in 15% of diffuse large B-cell (DLBC) lymphomas. In this study, 80% of the c-MYC rearrangements were detected in aggressive diffuse lymphoma subsets but, interestingly, 9% of FCL showed involvement of t(8q24) translocation. In our study, rearrangements of the BCL-6 gene at band 3q27 were found in 31% of DLBC lymphomas. Interestingly, 50% of the BCL-6 rearrangement positive lymphoma cases had coexisting gene rearrangements involving all of the aforementioned gene loci. The molecular dissection of these genes will improve our understanding of the genesis of the diverse clinicopathological subtypes.     

New recurrent structural chromosome aberrations in non-Hodgkin's lymphoma

Identification, and subsequent molecular dissection, of recurring structural chromosome aberrations has led to a substantial increase in our understanding of lymphomagenesis. Thus we have reviewed the published literature on cytogenetic findings in non-Hodgkin's lymphoma (NHL) in search of previously unrecognized recurring chromosome aberrations. Thirty-four balanced rearrangements, including 32 reciprocal translocations and two inversions, and 25 unbalanced translocations, each observed in at least two different cases of NHL and previously unrecognized as recurring, have been ascertained. Among the 32 reciprocal translocations, 10 involved bands harboring one of the immunoglobulin (Ig) genes. In nine of these, the following bands or regions may be sites of putative oncogenes that are activated through juxtaposition to Ig loci: 1p35-36, 5q11, 6q21, 9p24, 12q13, 13q11, 15p11, 15q21-22 and 15q23-24. In one instance, t(21;22)(q22;q11), Ig lambda chain gene involvement is unlikely, because the t(21;22) has been identified in two NHLs of T-cell lineage. An additional four reciprocal translocations and one inversion affected the band 3q27, containing the BCL6/LAZ3 gene, and one of the following bands: 1q25, 3q12, 6p21, 7p13, 12p13. Three other reciprocal translocations had the breakpoint at 11q13 known to harbor the BCL1 gene. Among the 16 remaining balanced rearrangements, one translocation involved a band containing a gene for a T-cell receptor, i.e. 7q35. Almost all chromosomes in the human karyotype (except 3, 8, 20 and 21) were implicated in at least one of the 25 recurrent unbalanced translocations. The distribution of resulting chromosomal imbalances is highly nonrandom, however, because 17 translocations involved the long arm of chromosome 1 (1q) invariably resulting in partial trisomy of 1q. We suggest that these unbalanced translocations of Iq are best regarded as non-specific secondary abnormalities that may contribute to lymphoma progression.     

Analysis of c-myc, bcl-1 and bcl-2 translocations in human lymphoma by pulsed-field gel electrophoresis.

Translocations of the c-myc, bcl-2 and the putative bcl-1 oncogene are recurrent events in B-cell lymphoma. Since it is likely that the rearranged genes contribute to the malignant phenotype of the tumor cells, such oncogene translocation is of major interest. The molecular detection of translocations using conventional Southern hybridization analysis is complicated by the fact that translocation breakpoints are dispersed over large chromosomal regions. In order to overcome this problem we used pulsed-field gel electrophoresis (PFGE) to detect c-myc, bcl-2 and bcl-1 translocations in 29 lymph node biopsies. C-myc translocation could not be detected in this group, either with standard Southern analysis of PFGE. Translocations of the bcl-2 gene were detected by PFGE in 5 samples and the breakpoints were mapped in all cases to the third exon of bcl-2 by standard Southern analysis. Furthermore, we also found rearrangements of the bcl-1 locus in 3 samples. Mapping of the breakpoint failed in one of these cases, which strongly indicates the existence of a breakpoint outside the bcl-1 major breakpoint region. Thus, PFGE allows the rapid detection of translocations in human lymphomas within large stretches of DNA.     

Identification of novel cryptic translocations involving IGH in B-cell non-Hodgkin's lymphomas

Chromosomal rearrangements involving the immunoglobulin heavy chain gene (IGH) at 14q32 are observed in approximately 50% of patients with B-cell non-Hodgkin's lymphoma (NHL). The 5' end of the IGH gene is located within 8 kb of the telomeric repeats of 14q. Translocations involving the IGH locus and the telomeric band of a partner chromosome are difficult to identify, because most terminal bands of human chromosomes appear pale by conventional G-banding techniques. To determine whether there are cryptic translocations involving the IGH locus, we used dual-color fluorescence in situ hybridization (FISH) of 5' and 3' IGH genomic clones containing the variable sequences, or the J(H) and the 5' constant regions, respectively. We examined cells from 51 patients with B-cell NHL who had a normal karyotype (3 patients), clonal abnormalities not involving 14q32 (35 patients), or alterations of 14q32 other than recurring translocations, i.e., add(14)(q32) (13 patients). FISH detected 17 IGH translocations in 16 of 51 (31%) cases. Of the 13 cases with add(14)(q32), FISH identified the partner chromosome in 9 cases (69%; 3q27, 6 cases; 2p13, 19p13.3, and 18q21.3, 1 case each). Six of thirty-eight (16%) patients without visible alterations of 14q32 and 2 of 13 (15%) patients with an abnormality of one chromosome 14 had masked (5 patients) or cryptic IGH translocations (3 patients), involving 3q27 (3 patients), 5p15.3 (2 patients), 19p13.3 (3 patients), or 14q32 (1 patient; 1 patient had two rearrangements). We identified two novel, recurring, cryptic translocations: t(5;14)(p15.3;q32) (2 patients) and t(14;19)(q32;p13.3) (3 patients). In summary, FISH permitted the detection of cryptic or masked IGH rearrangements in approximately 20% of lymphoma cases without visible rearrangements of 14q32 analyzed retrospectively.     

Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin's lymphoma by interphase fluorescence in situ hybridization.

Structural alterations in 3q27 affecting the BCL6 locus are among the most frequent changes in B-NHL. The aim of the present study was to establish an interphase-FISH assay for the detection of all diverse BCL6 translocations in B-NHL. Two different approaches were tested, one using a PAC-clone spanning the major breakpoint region (MBR) of BCL6 (span-assay), and another using two BAC clones flanking the MBR (flank-assay). Interphase FISH with the span-assay detected the various BCL6 translocations in seven B-NHL cell lines. The dual-color flank-assay was evaluated in two laboratories independently: in normal controls, the cutoff level for false-positive signals was 2.6%, whereas the cutoff level for false-negatives in the seven cell lines was 7.5%. To test the feasibility of the FISH strategies, 30 samples from patients with B-NHL with cytogenetic abnormalities of 3q27 were evaluated with both assays. In 21 cases, the span-assay indicated a BCL6 rearrangement. In 18 of the 21 cases, the dual-color flank-assay confirmed the translocation including 12 different partner chromosomal loci. The three false-positive cases detected with the span-assay showed trisomy of chromosome 3 by cytogenetic analyses, and they were correctly classified as non-rearranged with the flank-assay. In summary, our FISH strategy using two differently labeled flanking BCL6 BAC probes provides a robust, sensitive, and reproducible method for the detection of common and uncommon abnormalities of BCL6 gene in interphase nuclei. The routine application of this assay to patients with B-NHL will allow the assessment of the diagnostic and prognostic significance of BCL6 rearrangements.     

Molecular analysis of breaks in BCL-1 proto-oncogene in B-cell lymphomas with abnormalities of 11q13

The t(11;14)(q13;q32) is a recurring translocation that occurs infrequently but non-randomly in B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, and multiple myeloma. The putative oncogene BCL-1 located at the chromosomal band 11q13 has been cloned previously from a B-cell CLL with t(11;14). We studied the molecular structure of the BCL-1 gene in eight B-cell NHL tumors that exhibited a break at 11q13. The cytogenetic changes in these tumors were t(11;14) in three, t(1;11;14)(q32;q13;q32) in two and dup(11)(pter----q23::11q13----ter) in three. The BCL-1 gene was found to have rearranged in two tumors. By Southern blot analysis of single and double digested DNA from placenta and from the tumors, we mapped the breakpoint in BCL-1 to a 0.5 kb Pst-I-HindIII restriction fragment which was approximately 2kb away from the sites of previously mapped breakpoints. Sequential hybridization of Southern blots of these tumors with different immunoglobulin probes (for J region, Cu, Su) and BCL-1 probe identified co-migrating fragments with Su probe after BamHI restriction digestion. These results demonstrate that translocation breaks in the BCL-1 gene are not clustered in a short stretch of DNA at 11q13, and that the translocation related breaks in the immunoglobulin heavy chain can occur outside the joining region. The implications of these observations to the genesis of chromosomal translocations during B cell development are discussed.     

Novel FISH probes designed to detect IGK-MYC and IGL-MYC rearrangements in B-cell lineage malignancy identify a new breakpoint cluster region designated BVR2.

Detection of translocations involving MYC at 8q24.1 in B-cell lineage malignancies (BCL) is important for diagnostic and prognostic purposes. However, routine detection of MYC translocations is often hampered by the wide variation in breakpoint location within the MYC region, particularly when a gene other than IGH, such as IGK or IGL, is involved. To address this issue, we developed and validated four fluorescence in situ hybridization (FISH) probes: two break apart probes to detect IGK and IGL translocations, and two dual-color, dual-fusion FISH (D-FISH) probes to detect IGK-MYC and IGL-MYC. MYC rearrangements (four IGK-MYC, 12 IGL-MYC and four unknown partner gene-MYC) were correctly identified in 20 of 20 archival BCL specimens known to have MYC rearrangements not involving IGH. Seven specimens, all of which lacked MYC rearrangements using a commercial IGH/MYC D-FISH probe, were found to have 8q24 breakpoints within a cluster region >350-645 kb 3' from MYC, provisionally designated as Burkitt variant rearrangement region 2 (BVR2). FISH is a useful ancillary tool in identifying MYC rearrangements. In light of the discovery of the distally located BVR2 breakpoint cluster region, it is important to use MYC FISH probes that cover a breakpoint region at least 1.0 Mb 3' of MYC.     

Molecular analysis of an unstable genomic region at chromosome band 11q23 reveals a disruption of the gene encoding the alpha2 subunit of platelet-activating factor acetylhydrolase (Pafah1a2) in human lymphoma.

A region of 150 kb has been analysed around a previously isolated, lymphoma associated, translocation breakpoint located at chromosome band 11q23. This balanced and reciprocal translocation, t(11;14)(q32;q23), has been shown to result in the fusion between chromosome 11 specific sequence and the switch gamma4 region of the IGH locus. The LPC gene, encoding a novel proprotein convertase belonging to the furin family, has been identified in this region. In order to characterize further the region surrounding the translocation, we have determined the detailed structure of LPC. Here we show that LPC consists of at least 16 exons covering 25 kb, and that there is a partial duplication, involving mobile genetic elements and containing LPC exons 13-17 in a tail-tail configuration at 65 kb downstream. Since the chromosomal breakpoint lay between these two structures, the intervening region was further analysed and shown to contain at least two unrelated genes. The previously known SM22 gene was localized close to the 3' tail of LPC. Furthermore, we identified the gene encoding the alpha2 subunit of platelet-activating factor acetylhydrolase (Pafah1a2) at the chromosomal breakpoint. The position of another previously identified breakpoint was also located to within the first intron of this gene. Altogether, our results give evidence of a genomic instability of this area of 11q23 and show that Pafah1a2 and not LPC is the gene disrupted by the translocation, suggesting that deregulated Pafah1a2 may have a role in lymphomagenesis.     

Analysis of c-myc, bcl-1 and bcl-2 Translocations in Human Lymphoma by Pulsed-Field Gel Electrophoresis

Translocations of the c-myc, bcl-2 and the putative bcl-1 oncogene are recurrent events in B-cell lymphoma. Since it is likely that the rearranged genes contribute to the malignant phenotype of the tumor cells, such oncogene translocation is of major interest. The molecular detection of translocations using conventional Southern hybridization analysis is complicated by the fact that translocation breakpoints are dispersed over large chromosomal regions. In order to overcome this problem we used pulsed-field gel electrophoresis (PFGE) to detect c-myc, bcl-2 and bcl-1 translocations in 29 lymph node biopsies.

C-myc translocation could not be detected in this group, either with standard Southern analysis or PFGE. Translocations of the bcl-2 gene were detected by PFGE in 5 samples and the breakpoints were mapped in all cases to the third exon of bcl-2 by standard Southern analysis. Furthermore, we also found rearrangements of the bcl-1 locus in 3 samples. Mapping of the breakpoint failed in one of these cases, which strongly indicates the existence of a breakpoint outside the bcl-1 major breakpoint region. Thus, PFGE allows the rapid detection of translocations in human lymphomas within large stretches of DNA.     

Follicle center lymphoma is associated with significantly elevated levels of BCL-6 expression among lymphoma subtypes, independent of chromosome 3q27 rearrangements.

The BCL-6 gene, located on chromosome 3q27, is implicated in the normal germinal center formation and is frequently rearranged in a wide spectrum of lymphomas. However the links between genetic alterations and expression of the gene are not clearly determined. We established a quantitative RT-PCR assay based on TaqMan technology to quantify BCL-6 mRNA expression in different subtypes of lymphomas and to compare the level of expression in lymphomas characterized by the presence or absence of BCL-6 translocation. Total RNA was extracted from 105 nodes biopsies (35 diffuse large B cell lymphomas (DLBCL); 26 follicle center lymphomas (FCL); 7 marginal zone lymphomas (MZL); 6 mantle cell lymphomas (MCL); 6 chronic lymphocytic leukemia (CLL); 5 T cell lymphomas (TCL); 7 classical Hodgkin diseases (HD); 6 nodal metastasis (NM); and 7 reactive hyperplasia (RH)). BCL-6 gene rearrangement was assessed by Southern blot analysis in 75% of 3q27(+) DLBCL (n = 20) cases and 67% of 3q27(+) cases (n = 10). The highest level of relative BCL-6 expression was observed in FCL (9.12 +/- 7.28) comparatively to the other lymphoma subtypes including DLBCL (2.53 +/- 1.82; P < 0.001), MCL (1.23 +/- 0.73), MZL (1.49 +/- 1.3), HD (1.60 +/- 1.00), TCL (1.75 +/- 1.64), but also RH (3.91 +/- 3.12) or NM (1.95 +/- 2.6). Among the 26 FCL cases, we observed a lower expression in grade 3 (n = 8) than in grade 1/2 (P < 0.001). Conversely, we failed to show any difference between 3q27(+) DLBCL and 3q27(-)DLBCL cases (P = 0.42). Paradoxically BCL-6 expression in 3q27(+) FCL (n = 10) was significantly lower than in 3q27(-) FCL cases (P = 0.035). Finally, this study showed that BCL-6 expression in lymphoma is largely independent of chromosome 3q27 rearrangement and is more related to the histological subtype. Clinical implication and alternative deregulation pathways of BCL-6 expression remain to be determined.     

Prognostic impact of chromosomal alteration of 3q27 on nodal B-cell lymphoma: correlation with histology, immunophenotype, karyotype, and clinical outcome in 329 consecutive patients

Rearrangements involving the BCL6 gene are found in 30% of diffuse large B-cell lymphomas (DLBCLs). We evaluated the clinical characteristics and prognoses of patients with B-cell lymphoma carrying 3q27 translocations. Among the 59 patients having 3q27 translocation, 10.9% had follicular lymphoma (FL) and 23.1% had DLBCL. It is of interest that the prognostic significance was not found between FL and DLBCL with 3q27 translocations. Progression-free survival (PFS) rate was significantly higher in the FL patients with 3q27 translocation than in those with 18q21 translocation. PFS rate was significantly higher in the DLBCL patients with 3q27 translocation than in those with 18q21 translocation. These findings suggest that the presence of 3q27 translocation is a significant prognostic factor in DLBCL.     

Rearrangements of chromosome band 1p36 in non-Hodgkin's lymphoma.

We studied 850 consecutive cases of histologically ascertained pretreatment non-Hodgkin's lymphoma with cytogenetically abnormal clones. The diagnostic karyotypes revealed that 12% of these cases exhibited structural rearrangements involving chromosome band 1p36. Here, we describe the karyotypes of 53 cases containing a 1p36 rearrangement [often involving translocations of unknown material and presented as add(1)(p36)]. We used fluorescence in situ hybridization to determine the origin of the translocation partners. We report three different recurrent translocations involving 1p36. These include der(1)t(1;1)(p36;q21) (three cases), der(1)t(1;1)(p36;q25) (three cases), and der(1)t(1;9)(p36;q13) (four cases). Using cytogenetic and fluorescence in situ hybridization analyses, we have resolved the translocation partners in 31 cases. Rearrangements of band 1p36 were found among different histopathological subtypes. Alterations of 1p36 never occurred as a sole abnormality, and in 42 of 53 cases, alterations of the band 14q32 were observed. The t(14;18)(q32;q21) translocation was present in 35 cases. The significantly high occurrence of 1p36 breakpoint in structural rearrangements and its involvement in recurrent translocations suggest that the region is bearing gene(s) that are important in lymphomagenesis. Our study also showed that cytogenetically evident deletions were frequent in chromosome 1p, almost always involving the p36 region, whereas duplications were rare and never encompassed the p36 region. Chromosome band 1p36 harbors many candidate tumor suppressor genes, and we propose that one or more of these genes might be deleted or functionally disrupted as a molecular consequence of the rearrangements, thus contributing to lymphomagenesis.     

Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia

A region on chromosome 14q32.1 is often involved in chromosomal translocations and inversions with one of the T-cell receptor loci in T-cell lymphoproliferative diseases. The breakpoints of the different rearrangements segregate into two clusters; a cluster due to inversion on the centromeric side and a cluster due to simple balanced translocations on the telomeric side. If the target gene activated by these different types of chromosomal rearrangements is the same, the gene must be localized between the two clusters of breakpoints in a region of around 160 kb. Within this breakpoint cluster region, we isolated two genes; namely, TCL1 and TML1/TCL1b genes. In the course of characterizing the TML1 gene, we further identified a third novel gene, which we named TCL6 (T-cell leukemia/lymphoma 6), from a region 7 kb upstream of the TML1 locus. The TCL6 gene expressed at least 11 isoforms through very complex alternative-splicing, including splicing with the TML1 gene. Those isoforms encode at least five open reading frames (ORFs) with no homology to known sequences. The localization of the proteins corresponding to these ORF was determined by fusing green fluorescence protein at the carboxyl terminal of each ORF. ORF141 and ORF72 were observed in the cytoplasmic region, while ORF105, ORF119, and ORF163 were predominantly localized in the nuclear region. Since the TCL6 gene was expressed in T-cell leukemia carrying a t(14;14)(q11;q32.1) chromosome translocation and was not expressed in normal T-cells (just like the TML1 and TCL1 genes), it is also a candidate gene potentially involved in leukemogenesis. Oncogene (2000).     

Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas

Thyroid adenomas belong to the cytogenetically best investigated human epithelial tumors. Cytogenetic studies of about 450 benign lesions allow one to distinguish between different cytogenetic subgroups. Two chromosomal regions, that is, 19q13 and 2p21, are frequently rearranged in these tumors. Although 2p21 aberrations only account for about 10% of the benign thyroid tumors with clonal cytogenetic deviations, 2p21 rearrangements belong to the most common cytogenetic rearrangements in epithelial tumors due to the high frequency of these benign lesions. The 2p21 breakpoint region recently has been delineated to a region of 450 kbp, but the gene affected by the cytogenetic rearrangements still has escaped detection. Positional cloning and 3' RACE-PCR allowed us to clone that gene which we will refer to as thyroid adenoma associated (THADA) gene. In cells from two thyroid adenomas characterized by translocations t(2;20;3) (p21;q11.2;p25) and t(2;7)(p21;p15), respectively, we performed 3'-RACE-PCRs and found two fusions of THADA with a sequence derived from chromosome band 3p25 or with a sequence derived from chromosome band 7p15. The THADA gene spans roughly 365 kbp and, based on preliminary results, encodes a death receptor-interacting protein.