Oncogene rearrangements in chronic B-cell leukemia

Forty-four B-chronic lymphocytic leukemias (CLL) were studied by Southern blot analysis using probes for the Ig genes and bcl-1, bcl-2 (major, minor and 5' breakpoint region), bcl-3, c-myc, and retinoblastoma (Rb) loci. Eight cases had three or more rearranged JH bands, indicating oligoclonality, clonal evolution, or chromosomal translocation. One case had a rearrangement of the bcl-1 locus and three of the bcl-2 locus. In the first case, comigration of the rearranged bcl-1 and JH sequences indicated a t(11;14)(q13;q32) translocation, which, in contrast to previously described cases, seems to be completely reciprocal. One case with a bcl-2 rearrangement showed comigration of the bcl-2 major breakpoint region and a rearranged JH band. This indicates a t(14;18) (q32;q21). The two other cases showed rearrangements of the bcl-2 5' breakpoint region without apparent comigration. No rearrangements were detected of c-myc and bcl-3, located at chromosome 19, nor was a deletion of Rb found. All but three cases had CD5 expression. The exceptions included the t(11;14) and the t(14;18) cases. Our results confirm recent data on rearrangements at the 5' site of bcl-2 in CLL. Additionally, they corroborate the presumption that CD5-negative chronic B-cell leukemias should be considered apart from classical CLL.     

PRAD-1/cyclin D1 gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma

The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are frequently associated with mantle cell lymphomas (MCLs) and only occasionally with other variants of B-cell lymphoid malignancies. This translocation seems to activate the expression of PRAD-1/cyclin D1 gene located downstream from the major breakpoint cluster region of this rearrangement. However, the possible overexpression of this gene in other lymphoproliferative disorders independently of bcl-1 rearrangement is unknown. We have examined the overexpression of PRAD-1 gene in a large series of 142 lymphoproliferative disorders including 20 MCLs by Northern blot analysis. Cytogenetic and/or bcl-1 rearrangement analysis with 2 probes (MTC, p94PS) were performed in 28 cases. Strong PRAD-1 overexpression was observed in 19 of the 20 MCLs including 3 gastrointestinal forms and 4 blastic variants. t(11;14) and/or bcl-1 rearrangement was detected in 6 of the 12 MCLs examined. No correlation was found between the different levels of mRNA expression and the pathologic characteristics of the lymphoma. Among chronic lymphoproliferative disorders other than MCL, only 1 atypical chronic lymphocytic leukemia (CLL) with a t(11;14) translocation and bcl-1 rearrangement and the 2 hairy cell leukemias (HCLs) analyzed showed upregulation of PRAD-1 gene. The expression in the 2 HCLs was lower than in MCL, and no bcl-1 rearrangement was observed. These findings indicate that PRAD-1 overexpression is a highly sensitive and specific molecular marker of MCL but it may also be upregulated in some B-CLLs and in HCL.     

Association of bcl-1 rearrangements with lymphocytic lymphoma of intermediate differentiation

Previous studies using classical cytogenetics have demonstrated the presence of the t(11;14) (q13;q32) chromosomal translocation in some cases of lymphocytic lymphoma of intermediate differentiation (IDL), a distinct type of low grade B-cell lymphoma. This finding suggested that the bcl-1 region (located at band q13 of chromosome 11) might be involved in this neoplasm. Using a genomic probe from the major breakpoint area of the bcl-1 locus, we identified rearrangements of the bcl-1 region in 10 of 19 cases, 2 of which comigrated with a rearranged allele of the immunoglobulin heavy chain gene joining region. In contrast, bcl-1 rearrangements were not found in other types of low grade B-cell lymphoma, specifically in 36 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and 27 cases of follicular lymphoma (FL). To further assess the molecular pathology of IDL, we analyzed these cases for rearrangements of the bcl-2 proto-oncogene, which is associated primarily with follicular lymphomas. None of the 19 cases of IDL had rearrangements. Furthermore, none of the 36 cases of CLL/SLL showed bcl-2 rearrangements, whereas, as expected, 21 of 27 cases of FL had rearrangements of the bcl-2 locus. Our findings demonstrate an association between a rearranged bcl-1 region with approximately 50% of IDLs and suggest that abnormalities of this locus may be important in the pathogenesis of IDL.     

Molecular analysis of bcl-1/IgH junctional sequences in mantle cell lymphoma: potential mechanism of the t(11;14) chromosomal translocation

Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation that juxtaposes the bcl-1 locus to immunoglobulin (Ig) gene sequences and leads to deregulation of cyclin D1 gene. t(11;14) is thought to result from an error of the recombinase during D-JH Ig gene assembly; however, data on the underlying mechanism and candidate recombination-targeting motifs in the major translocation cluster (MTC) of the bcl-1 gene are lacking. bcl-1/IgH junctional sequences from seven MCL patients were amplified by PCR using primers targeting MTC and JH sequences on chromosomes 11q13 and 14q32, respectively. PCR products were directly sequenced and junctional sequences were searched for homology to known germline D genes. bcl-t MTC breakpoints were searched for the presence of possible recombination target motifs; heptamers, nonamers, binding sequence of the bp45 nuclease, x-like sequences and D gene segments. bcl-1/JH junctions were found to bear homology to D gene segments (DLR3, DM and DIR5) in 3/7 MCL samples. A computer-based search in previously published and/or submitted to GenBank bcl-1/JH junctional sequences identified homology to D genes in 1/4 MCL tumour samples and 1/4 MCl cell lines; DXP4 or D23/7 and DHQ52 or D22/21 or DXP5, respectively. The MTC locus contained motifs with homology to bp45 nuclease binding sequence, x-like sequences, heptamers/nonamers, D-like DIR genes and non-homologous recombination short (6 bp) DNA sequences. The above data indicate that the t(11;14) translocation in MCL may also occur at a more mature stage of B-cell ontogeny than previously thought, i.e. during VH-to-DJH rearrangement. Various known recombination motifs within MTC may contribute to an illegitimate recombination event between bcl-1 and DJH.     

B-CLL and bcl-2 gene

Most of human follicular lymphomas (approximately 90% in U.S.A. or approximately 30% in Japan) possess the t(14; 18) chromosome translocation that directly involves the IgH locus on chromosome 14 and the bcl-2 gene on chromosome 18. The t(14; 18) chromosome translocation occurs nearly exclusively at two hot spots, a major breakpoint clustering region (mbr) within the 2nd exon noncoding region and the minor breakpoint clustering region (mcr) within the 3' flanking region of the bcl-2 gene. Here we show that the rearrangement of the bcl-2 gene occurs in a significant fraction (approximately 10%) of B-CLL. All of the rearranged bcl-2 genes were juxtaposed with Ig lambda or Ig kappa genes, implying that the bcl-2 gene is preferentially linked to the IgL genes in CLL.     

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.     

Nuclei micro-array FISH,a desirable alternative for MCL diagnosis

Mantle cell lymphoma (MCL) is a rare, specific lymphoma subtype. Though the morphologic and immunophenotypic features of MCL have been well described in recent literatures, it is still a diagnostic dilemma because of its frequent confusion with other small B cell lymphomas (SBCLs). In the present study, we primarily focus on establishing a sensitive and specific method for the diagnosis of MCL, which is efficient to distinguish this disease from other SBCLs. We carried out our investigation for MCL and other SBCLs (including SLL, FL, MZL, and MALT) on their feature of morphology, immunophenotype, and t(11;14)(q13;q32) translocation analysis based on polymerase chain reaction (PCR) and interphase nuclei micro-array fluorescence in situ hybridization (FISH). The morphologic and immunologic analysis showed the positive rate of cyclin D1 was 76.47% in MCL, which was significantly higher than that in other SBCLs. The positive rate of t(11;14) translocation was 25.81% and 35.48%, respectively, tested by general and semi-nested PCR, while 93.10% positive rate was shown with low background and strong signals pattern when tested by Nuclei micro-array FISH. Our research shows that t(11;14) translocation is a special and useful diagnostic marker for MCL, and detection of the marker by nuclei micro-array FISH is convenient and economic, especially more sensitive and specific than other methods for the diagnosis of MCL.     

Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization

Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.     

Correlation of bcl-2 rearrangement with clinical characteristics and outcome in indolent follicular lymphoma

The t(14;18) translocation, which involves the bcl-2 oncogene, occurs in follicular lymphomas (FL) at two common sites: the major breakpoint region (MBR) and the minor cluster region (mcr). The biological and clinical significance of these breakpoints is unknown. The bcl-2 breakpoint site was determined in 247 previously untreated patients (49% men; median age 52 years) with indolent FL (155 grade I, 83 grade II, and 8 grade III) to correlate it with pretreatment characteristics, response, and outcome. The bcl-2 breakpoint site was determined by a polymerase chain reaction method of peripheral blood (all cases), bone marrows (149 cases), and fresh lymph node biopsy specimens (68 cases). The breakpoint site occurred at MBR in 175 cases (71%) and at mcr in 27 (11%). In 45 cases (18%), no breakpoint was detected (germline). No significant relationship was found between the rearrangements and the expression of BLC-2 and BAX proteins. Patients' germline for MBR and mcr tended to present more frequently with stage IV disease and higher beta2-microglobulin (beta2M) levels, whereas mcr-rearranged patients presented more frequently with early stage and normal beta2M. The complete response rate of germline patients was significantly lower than that of MBR and mcr patients. An estimated 3-year failure-free survival (FFS) for mcr, MBR, and germline cases was 95%, 76%, and 57%, respectively (P <.001). The bcl-2 breakpoint site was independent of serum beta2M and lactate dehydrogenase in its correlation with FFS. In conclusion, the bcl-2 rearrangement site is an important prognostic factor in indolent FL, useful to identify patients who may require different treatment.     

Small lymphocytic lymphoma/chronic lymphocytic leukemia with t(11;14)]

A 83-year-old woman was referred to our hospital because of swollen lymph nodes, marked splenomegaly, and bone marrow abnormality. Histological examination of the lymph nodes revealed characteristic findings for small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The immunophenotype of the tumor cells was CD5+, 10-, 19+, 20+, 23-, IgM+D+. Interphase fluorescent in situ hybridization (FISH) detected t(11;14), and immunohistochemical studies demonstrated cyclin D1 expression. In both SLL/CLL and mantle cell lymphoma (MCL), the normal counterpart of the tumor cells is thought to be CD5-positive B1 cells. The present case may therefore have been borderline between SLL/CLL and MCL.     

Diagnostic utility of fluorescence in situ hybridization in mantle-cell lymphoma

Mantle-cell lymphoma (MCL) has a poorer prognosis than other small B-cell lymphomas, thus a definitive diagnosis is essential. The t(11;14)(q13;q32) associated with MCL juxtaposes portions of CCND1 (11q13) and IGH (14q32), resulting in over-expression of cyclin D1. In this study, a highly sensitive two-colour fluorescence in situ hybridization (FISH) method was developed to detect t(11;14)(q13;q32) in nuclei isolated from paraffin-embedded tissue. Twenty-three MCLs, 13 normal controls and nine small B-cell lymphomas other than MCL were studied by FISH. Each MCL had been previously investigated to detect genomic IGH-CCND1 fusion by polymerase chain reaction (PCR) using DNA extracted from frozen tissue. The IGH-CCND1 fusion detection rate in the MCLs was 96% by FISH compared with 35% by PCR. By FISH, one MCL and three small B-cell lymphomas other than MCL harboured abnormalities involving only IGH. Less than 1% of cells showed false-positive IGH-CCND1 fusion in normal specimens by FISH. Thus, this highly sensitive FISH assay is very useful in confirming the diagnosis of MCL, has wide applicability as it may be performed on both paraffin-embedded and fresh tissue, and may also facilitate detection of translocations involving these loci in tumours other than MCL.     

Detection of the chromosomal translocation t(11;14) by polymerase chain reaction in mantle cell lymphomas

The t(11;14)(q13;q32) and its molecular counterpart, BCL1 rearrangement, are consistent features of mantle cell lymphoma (MCL). Rearrangement is thought to deregulate the nearby CCND1 (BCL1/PRAD1) proto-oncogene, a member of the cyclin G1 gene family, and thereby to contribute to tumorigenesis. We and others have previously shown that the BCL1 locus is rearranged in 55% to 60% of MCL patients and that, on chromosome 11, more than 80% of the breakpoints are localized within a 1-kbp DNA segment known as the major translocation cluster (MTC). We have determined the nucleotide sequence for a portion of the MTC region, and constructed chromosome 11-specific oligonucleotides that were in conjunction with a consensus immunoglobulin (Ig) heavy chain joining region (JH) primer used to perform the polymerase chain reaction (PCR) to amplify t(11;14) chromosomal junctional sequences in DNA from 16 MCL patients with breakpoints in the MTC region. 15 of the 16 breakpoints that occurred at the MTC region were amenable to PCR detection. The sizes of the amplified bands, the existence or not of a Sac I site in the PCR products, and nucleotide sequencing of the amplified DNA from four patients showed that the breakpoints share a remarkable tendency to tightly cluster within 300 bp on chromosome 11, some of them occurring at the same nucleotide. On chromosome 14, the breakpoints were localized within the Ig JH. Our findings indicate that a BCL1 rearrangement can be detected using this approach in roughly one half of the MCL patients. This has implications for both the diagnosis and the clinical management of MCL.     

Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region

A small (2.8-kilobase, kb) major breakpoint region localized to segment 18q21 rearranges in greater than 70% of t(14;18)(q32;q21) lymphomas. This rearrangement interrupts the Bcl-2 gene and introduces it into the Ig locus at 14q32. The rearrangement between the joining region (JH) of Ig on chromosome 14 and the 18q21 region creates a translocation- specific DNA rearrangement. We generated probes that distinguish the 14;18 juncture on the derivative (der) 14 and der (18) chromosomes, providing a molecular approach to t(14;18) identification. Approximately 60% of unselected follicular lymphomas, 20% of diffuse large cell lymphomas, and 50% of adult undifferentiated non-Burkitt lymphomas demonstrated 14;18 rearrangements within the major breakpoint region. Examination of DNA for 14;18 rearrangements resolved the identity of 14q+ chromosomes in two patient's cells that lacked an obvious reciprocal partner. Identification of the exact restriction fragments that mediate translocations complements routine cytogenetics. The detection of DNA rearrangements does not require dividing cells or the presence of an identifiable reciprocal partner and can detect clonal translocation rearrangements when the neoplastic cells are only a minority of all cells present.     

p53 overexpression as a marker of poor prognosis in mantle cell lymphomas with t(11;14)(q13;q32)

The t(11;14)(q13;q32) translocation, which juxtaposes the BCL1 oncogene with the Ig heavy chain locus, has been associated with an uncommon subtype of non-Hodgkin's lymphoma (NHL) termed mantle cell lymphoma (MCL). To date, no molecular marker that serves as an indicator of tumor progression or clinical prognosis has been described for NHLs with this translocation. We examined a panel of NHLs with t(11;14) for overexpression of p53 and correlated the results with single-strand conformation polymorphism (SSCP) analysis, karyotypic features, and clinical course. NHLs with t(11;14) were identified from 30 patients. The diagnosis was MCL for 23 of 30, small lymphocytic lymphoma for 4 of 30, and diffuse large-cell lymphoma for 3 of 30 cases. The results of immunohistochemistry analysis using a monoclonal anti-p53 antibody on paraffin-embedded specimens were compared with the SSCP data, the tumor karyotypes, and clinical course of each patient. DNA sequencing of exons was performed on cases that showed conformational changes by SSCP analysis. NHLs from 5 of 23 patients with MCL were positive for p53 overexpression. Deletions of chromosome 17p were identified in 2 of 30 cases, both of which were MCLs showing p53 overexpression. Two of the five MCLs with p53 overexpression showed evidence for TP53 mutations. None of the 18 MCLs negative for p53 overexpression showed conformational changes by SSCP. For these 18 patients with MCLs that did not overexpress p53, the median survival was 63 months, compared with 12 months for the 5 patients with MCLs positive for p53 overexpression (P < .001). These results suggest that p53 overexpression in MCL with t(11;14)(q13;q32) may serve as a marker of poor prognosis.     

Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas

A majority of t(14;18) translocations have been shown to cluster at one of two sites on chromosome 18, called the major breakpoint region (mbr) or the minor cluster region, (mcr), which map within or flanking the bcl-2 proto-oncogene, respectively. We have determined the nucleotide sequence for a portion of the mcr, and constructed oligonucleotides that were used to perform the polymerase chain reaction (PCR) in conjunction with universal immunoglobulin primers to specifically amplify t(14;18) breakpoints in DNA obtained from follicular lymphomas. Eight of ten breakpoints that were detectable on Southern blots using DNA probes for the mcr could be detected due to specific amplification by the PCR technique using an mcr-specific primer. Direct nucleotide sequencing of the enzymatically amplified DNAs showed that the breakpoints clustered within a 500 nucleotide region, and five occurred within three nucleotides of each other. These data show a remarkable clustering of some t(14;18) breakpoints at a site on chromosome 18, at least a 30-kb distance from the bcl-2 gene. Our findings also indicate that mcr-specific primers may be used in conjunction with previously described mbr-specific primers in a highly sensitive DNA amplification technique to detect a large fraction of t(14;18) breakpoints.     

The bcl-2 oncogene in Hodgkin's disease arising in the setting of follicular non-Hodgkin's lymphoma

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.     

Genetics of small lymphocyte disorders.

Cytogenetic analysis of small lymphocytes disorders is hindered by the low mitotic activity of the malignant cells. The use of fluorescence in situ hybridization (FISH) allows the detection of chromosomal amplifications, deletions, or translocations at a single-cell level in dividing and resting cells. The use of FISH in combination with other molecular techniques has defined the deletion in band 13q14 as the most common abnormality in chronic lymphocytic leukemia, followed by del (11)(q22-23), trisomy 12, del (17)(p13), and del (6)(q21). The del 13q14 is also found in 70% of mantle-cell lymphomas (MCLs) and in non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) patients. These findings point to the existence of yet unidentified tumor-suppressor gene(s) at the 13q14 locus, the loss/inactivation of which leads to B-cell neoplasia. Del (17(p13) (involving the p53 tumor-suppressor gene) and del (11)(q22-23) (involving the ataxia-telangiectasia gene [ATM]) seem to be independent prognostic factors for poor survival in chronic lymphocytic leukemia (CLL) patients. In MCL, the t(11;14) involving the bcl-1 gene is found, but data from a bcl-1 transgenic animal model suggest that hyperexpression of bcl-1 is not sufficient for lymphomatogenesis. Similar data are observed in bcl-2 transgenic animals, a finding showing that the bcl-2 hyperexpression observed in t(14;18)-positive follicular lymphoma cells is not sufficient to confer a malignant phenotype. The contribution of other chromosomal abnormalities other than bcl-1 and bcl-2 rearrangements in the pathogenesis of MCL and follicular-cell lymphomas has to be determined.     

Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas

Follicular lymphomas comprise almost two thirds of the US adult non- Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR- amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.     

BCL6 rearrangement in a patient with mantle cell lymphoma

 We describe a patient with mantle cell lymphoma (MCL) associated with BCL6 gene rearrangement. MCL is a distinct subtype of non-Hodgkin's lymphoma characterized by CD5+, CD10–, CD20+, t(11;14)(q13;q32) and PRAD1/cyclin D1 overexpression. Although rearrangement of the BCL6 gene is the most frequent genetic change among diffuse lymphomas and some follicular lymphomas this is the first report of a patient with MCL associated with BCL6 rearrangement. Received: 6 January 1997 / Accepted: 17 February 1997