Frequency and structure of t(14;18) major breakpoint regions in non-Hodgkin's lymphomas typed according to the Kiel classification: analysis by direct DNA sequencing.

We have examined 165 unselected cases of non-Hodgkin's lymphomas for rearrangements involving the t(14;18) major breakpoint region using a polymerase chain reaction (PCR) and direct sequencing of amplified major breakpoint region bcl-2/JH junctional regions. The lymphomas, diagnosed according to the updated Kiel classification, consisted of 33 centroblastic-centrocytic, 37 centroblastic, 27 immunocytic, 10 immunoblastic, 10 centrocytic, 2 lymphoblastic, 2 Ki-1-positive anaplastic large cell, 14 peripheral T-cell, and 4 unclassified lymphomas. In addition 18 chronic lymphocytic leukemias, 2 hairy cell leukemias, and 6 plasmacytomas were studied. In 17 cases a bcl-2/JH gene fusion sequence was amplified by PCR. A bcl-2/JH gene fusion was detected only in three lymphoma subgroups: 13 of 33 centroblastic-centrocytic (39%), 2 of 37 centroblastic (6%), and 2 of 27 immunocytic (8%) were positive. In two cases, major breakpoint region bcl-2 rearrangements verified by genomic Southern analysis were not detected by PCR. Direct sequencing of all 17 PCR-amplified, previously uncharacterized t(14;18) junctional regions provided corroborating evidence for the specificity of the assay. The procedure gave sequencing results even from limited amounts of lymphoma cells as obtained by fine needle aspiration of lymph nodes or from clinically uninvolved sites. Clone-specific sequences were identified due to the involvement of different JH segments, the variations among the exact JH and bcl-2 breakpoint positions, and the extensive incorporation of junctional region (D-) N-nucleotides. These clone-specific sequences allow accurate identification of clinically occult lymphoma cells and reduce the threat of false positive results. The finding of exceptionally long intervening sequences in some of the junctions and the partial homology with published DH segments in three cases support the view that some of the putative N-regions harbor DH regions.

Use of UITma DNA polymerase improves the PCR detection of rearranged immunoglobulin heavy chain CDR3 junctions.

The development of rapid PCR protocols for amplification of rearranged IgH gene sequences has greatly facilitated the identification of clonal IGH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias. However, the 15-35% incidence of false negative results with this approach has been a constant and unresolved problem. To assess the reliability of a previously published framework region 3 (FR3A) IgH-CDR3-PCR for detection of monoclonal IgH gene rearrangements we compared the PCR and Southern results in a series of 44 NHL and leukemias of B cell lineage showing a JH-rearrangement in Southern analysis with genomic DNA and hybridization with a IgH joining region (JH) probe. IgH-CDR3 regions were amplified using DNA extracted from clinical specimens by PCR using fluorescent dye-labeled consensus primers homologous to conserved regions within the variable (VH) and the joining (JH) gene segments. The PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. With commonly used DNA polymerases monoclonal IgH-CDR3 junctions were identified in 36/44 samples (82%). However, in the remaining eight cases (18%) with pathohistologically clearly demonstrated B cell malignancies which were also monoclonal on JH-Southern analysis, monoclonality could be demonstrated by FR3A-IgH-CDR3-PCR only with the proofreading UITma DNA polymerase. In four of these monoclonal VH--N--DH--N--JH junctions sequence analysis was performed which showed a point mutation in one and a single nucleotide deletion at the 3' terminus of the primer target site in the other case. In the remaining two cases no primer mismatches could be identified. Thus we conclude that the marked improvement of the PCR-detection rate of monoclonal IgH-CDR3 junctions was achieved at least in part due to the ability of UITma DNA polymerase to remove mismatched bases at the 3' terminus of the primers with respect to the target during the first amplification cycles. Our results suggest, that UITma is the DNA polymerase of choice for amplification of IgH-CDR3 junctions with consensus FR3A-VH- and JH-primers.

Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR.

PCR of clonally rearranged immunoglobulin heavy chain (IgH) gene sequences is increasingly used for detection of minimal residual disease (MRD) in lymphoid malignancies. Inherent quantitating problems are the main drawbacks of traditional PCR technologies. These limitations have been overcome by the recently developed real-time quantitative PCR (RQ PCR) technology. However, clinical application of the few published RQ PCR assays targeting immune gene rearrangements is hampered by the expensive and time-consuming need for individual hybridization probes for each patient. We have developed a new RQ PCR strategy targeting clonally rearranged IgH sequences that solves this problem. The method uses only two different JH hybridization probes and four downstream JH primers homologous to consensus germline JH gene segments. In combination with an allele-specific upstream (ASO) primer the consensus JH probes and primers allow quantitation of about 90% of possible IgH rearrangements. In a series of 22 B-lineage ALL the new assay allowed the detection of one to 10 blasts in a background of 10(5) normal cells. To prove the clinical utility we quantified MRD in 23 follow-up samples of six ALL patients with the new assay in comparison with a published RQ PCR technique that used individually designed primer/probe sets. We showed that the sensitivity of the new RQ PCR assay was slightly higher for four of the six cases and about 100-fold higher for one case, enabling detection of an increasing MRD level as an indicator of subsequent relapse 44 weeks earlier compared to the ASO probe assay in this particular patient. The results suggest, that the novel RQ PCR assay is a rapid, technically simple, reliable, and sensitive alternative to traditional quantification assays and simplifies current approaches of monitoring MRD in clinical trials.

Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.

The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas.

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.

Polymerase Chain Reaction Analysis of t(14;18) Junctional Regions in B-Cell Lymphomas.

The polymerase chain reaction (PCR) procedure was used for rapid and highly specific amplification of the t(14;18) bcl-2/JH DNA junctional regions in B-cell lymphomas. By using Taq-polymerase and relatively long oligonucleotide primers-a 33-mer for bcl-2 and an universal 25-mer complementary to the JH consensus sequence-the primer annealing and primer extension steps could be carried out at the same temperature (70°C), thus markedly reducing the reaction time and significantly improving the specificity of the reaction. The specificity of the amplification allowed visual identification of the bcl-2/JH PCR-products in ethidium bromide stained agarose gels. DNA-sequence analysis of PCR-amplified, previously uncharacterized t(14; 18) junctional regions, confirmed the specificity of this assay. Moreover, preliminary data show that the procedure is capable of documenting the presence of occult lymphoma cells in both the peripheral blood and bone marrow.

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer.

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS--PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis.

Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific markers are increasingly being used in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) patients instead of the time consuming and labor intensive Southern analysis. In previous reports, no single common V beta and J beta sequence had been identified that allowed reliable amplification of the majority of rearranged T-cell antigen receptor (TCR)-beta V-D-J junctions at the DNA level because of the relatively large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes. In the present study we designed highly degenerate PCR primers directed against conserved sequences of the J beta genes. IN combination with a previously published consensus V beta primer, these J beta primers specifically amplify TCR- beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patients with T-cell lymphoproliferative disorders, one c-ALL patient, and five patients with nonmalignant diseases. T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls. Individual PCR products were sequenced after cloning. The nucleotide sequences of 96 randomly chosen recombinant vectors were determined. In the polyclonal controls all analyzed clones differed in their TCR-beta V-N(D)N-J junctions. In the T-cell lines, in all of the T-cell malignancies, and in the c-ALL, monoclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions. The PCR results were confirmed by automated fluorescence quantification and size determination of PCR products after separation in a high-resolution polyacrylamide gel. The procedure allows rapid and specific characterization of clonal TCR-beta rearrangements from genomic DNA and will significantly simplify current experimental approaches to identify and to quantitate malignant T cells during initial staging and follow-up of T-lineage NHL and ALL patients.

Sequence analysis of amplified t(14;18) chromosomal breakpoints in B-cell lymphomas.

We have explored different strategies for sequencing of major breakpoint (mbr) junctional regions in t(14;18) chromosomal translocations--the most frequent chromosomal abnormality observed in B-cell lymphomas. We demonstrate that coupling of the preparation of single-stranded DNA by asymmetric polymerase chain reaction (PCR) and direct sequencing is the method of choice for the rapid and precise determination of clone-specific bcl-2/JH fusion gene sequences. The rapidity, relative ease, and accuracy of the technique, described for the nucleotide sequence analysis of mbr t(14;18) breakpoints, permits the analysis of a relatively large number of samples and should be considered as part of the clinical evaluation of lymphoma patients. Furthermore, by providing sequence information of clone-specific DNA regions, the procedure should reduce the risk of false-positive results from PCR.

Characterization of clone-specific rearrangement T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing.

The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T-NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T-cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.

Clinical characteristics of high-grade lymphomas with immune genes in germline configuration.

In a prospective study of 42 high-grade lymphomas which were categorized according to the Kiel classification, the clinical significance of immune genotyping was studied. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were investigated. In 33 cases the immune genotype confirmed the phenotype. In one case with equivocal phenotype a TCR beta-chain rearrangement proved the T-cell origin of the lymphoma. None of the cases showed a bigenotype. There were eight lymphomas with immunoglobulin and TCR beta-chain and gamma-chain genes in germline configuration, which were divided into a group of immature lymphomas and a group of lymphomas with a more mature phenotype. The immature lymphomas had widespread disease, rapid progression, and favorable prognosis after intensive chemotherapy. The group of T-cell and Ki-1 lymphomas with null-cell genotype was clinically heterogeneous. Three of four cases were secondary lymphomas after lymphomatoid papulosis, lymphomatoid granulomatosis, or Hodgkin's disease. All cases presented with extranodal involvement. Only one of these patients is in continuous remission. In conclusion, the lack of immunoglobulin and TCR beta-chain and gamma-chain gene rearrangements does not exclude the diagnosis of high-grade malignant lymphoma, especially in cases with unusual extranodal involvement. However, the DNA analysis identifies a null-cell genotype subset of high-grade lymphomas which may have clinical significance.

Characterization of Raji cell binding IgG in patients with metastatic breast cancer.

The aim of the present study was to isolate and characterize the immune complexes detected by the Raji cell assay in metastatic breast cancer. However, the Raji cell binding material could not be separated from monomeric IgG by Sephacryl S-300 gel chromatography in any of the 16 sera investigated. The parallel elution profile of monomeric IgG and the Raji cell binding activity suggested that anti-Raji cell antibodies, rather than immune complexes of low molecular weight, were present in these sera. This was further substantiated by pepsin digestion of the Raji cell binding IgG fractions. The binding of F(ab')2 fragments was quantitatively comparable to the binding of undigested IgG, and the bound F(ab')2 fragments could be visualized by immunofluorescence at the cell membrane. The presence of antibodies against Raji cells was further confirmed by the complement-dependent cytotoxicity assay. These antibodies did not react with untransformed lymphocytes and there was no correlation with anti-Epstein-Barr virus antibodies. The incidence of cytotoxic anti-Raji cell antibodies in breast cancer was 12% compared to 20% in malignant lymphoma, to 0% in normals and patients with degenerative cardiovascular diseases, and to 5% in patients with auto-immune diseases.

[Rearrangements of immunoglobulin and T-cell-antigen receptor genes as diagnostic markers in lymphatic neoplasms]. / Rearrangements von Immunglobulin- und T-Zell-Antigenrezeptor-Genen als diagnostische Marker bei lymphatischen Neoplasien.

During recent years cloning of the genes encoding the immunoglobulin (Ig) and T-cell (TCR) antigen receptor has made it possible to analyze clonal expansions of B- or T-cells at the molecular level. We here describe the usefulness of the Ig- and TCR-gene rearrangements in selected cases of malignant lymphoma. In contrast to all other cases investigated, one non-Hodgkin's lymphoma (NHL) exhibited the infrequent phenomenon of oligoclonality, i.e. three distinct B-cell clones were discerned by Southern blot analysis. Detection of clonal TCR- and Ig-gene rearrangements unequivocally documented bone marrow involvement in four of eight NHL-patients, where conventional histologic and cytologic examination remained inconclusive. As expected, analysis of bone marrow DNA revealed clonal Ig-gene rearrangements in three cases with clear histologic evidence of bone marrow infiltrations by the NHL. In one case of a patient with acute mixed lymphoid-myeloid lineage leukemia a previously identified clonal Ig-gene rearrangement vanished after successful chemotherapy. Analysis of Ig- and TCR-gene rearrangements promises to be a very useful diagnostic tool in selected cases of lymphoid neoplasia.

Heterogeneity of immunoglobulin gene rearrangements in B-cell lymphomas.

We have examined 69 B-cell non-Hodgkin's lymphomas (NHL) for rearrangements of the immunoglobulin (Ig) or T-cell antigen receptor (TCR) genes. The lymphomas were assigned to the categories of the Kiel classification and their B-cell nature was confirmed by immunostaining. Only 2 cases (with CLL) displayed clonal T beta-chain TCR gene rearrangements together with rearranged heavy- and light-chain Ig genes. The remaining 67 lymphomas had a germline beta-chain TCR-gene configuration. Three different patterns of Ig gene rearrangements were identified; (A) presence of both heavy- and light-chain rearrangements (H+L+); (B) rearrangement of heavy-chain gene only (H+L-); (C) heavy- and light-chain genes in germline configuration (H-L-). All the 45 low-grade NHLs and the 4 immunoblastic lymphomas exhibited pattern A and all had their kappa gene rearranged or deleted. Of 24 low-grade lymphomas tested, 13 (54%) had an addition rearrangement of the lambda light-chain gene. In contrast, the 19 high-grade centroblastic (cb) B-NHLs had distinct patterns of Ig-gene rearrangement: 12 with pattern A, 4 with B and 2 with C. In this group only 2 of 17 (12%) cases analyzed had evidence of lambda light-chain rearrangement whereas 12 of 18 (67%) had a kappa gene rearrangement or deletion. In one case expressing sIgM/lambda and with heavy chain Ig-rearrangement, no DNA was available for Ig light-chain analysis.

Binding characteristics of three complement dependent assays for the detection of immune complexes in human serum.

The fluid phase C1q binding, the solid phase C1q binding and the Raji cell assay are the most widely employed methods for the detection of complement fixing immune complexes in human disease. However, their binding characteristics for complexes formed in native serum have not been compared and studied in detail. For this purpose, Tetanustoxoid (TT): anti-TT complexes were formed closely to in vivo conditions by incubating sera of immunized people with TT. Then the molecular characteristics of those complexes, which could be detected by the 3 immune complex assays, were defined. The methods used are precipitation curve, gel chromatography and measuring the complexed antibody with a sensitive Elisa method. All 3 assays detected only complexes near the equivalence zone; the Raji cell assay being the most sensitive detecting 1.5 micrograms complexed antibody/ml serum followed by the solid phase (6 micrograms/ml) and the fluid phase C1q binding assay (50 micrograms/ml). As estimated by gel chromatography, both the C1q binding assays measured most sensitively complexes with a molecular weight distinctly over 2,000,000 daltons, whereas the Raji cell assay detected preferentially complexes in the range of 2,000,000 daltons. Smaller TT: anti-TT complexes with a molecular weight less than 600,000 daltons were not detectable by the 3 assays. In conclusion, the 3 assays, while differing in sensitivity, showed similar binding patterns with preference for high molecular complexes near equivalence. Subsequently, they might be insensitive to small complexes persisting in the circulation.

Low incidence of mbr bcl-2/JH fusion genes in Hodgkin's disease.

Lymph node biopsies from 140 cases of Hodgkin's disease (HD) and from 30 non-malignant lesions were screened for the presence of t(14;18) translocations involving the major breakpoint region (mbr) of the bcl-2 gene and the joining region (JH) of the immunoglobulin heavy chain gene, using a polymerase chain reaction (PCR) assay with subsequent nucleotide sequencing of amplified bcl-2/JH junctional regions. Expression of the bcl-2 protein within the Hodgkin and Reed-Sternberg (HRS) cells was investigated in 86 cases of HD by immunohistochemistry on cryostat or paraffin sections. Although bcl-2 expression could be found in a proportion of neoplastic cells in up to one-third of HD cases, the frequency of t(14;18) gene fusions detected by PCR was low. We identified such gene fusions in only 3 out of 140 (2 per cent) HD cases, one biopsy of which presented with four clonally distinct bcl-2/JH sequences. No t(14;18) was found in any of 30 reactive lymph node lesions. All fusion gene sequences were unique regarding the localization of the chromosome 14 and 18 breakpoints and the extranucleotide N-insertions. None of these gene fusions conformed to t(14;18) breakpoint sequences previously characterized in our laboratories. Our findings point to a mere coincidence in some cases of HD lesions and cells carrying a t(14;18) in the same biopsy and argue against a significant role of bcl-2 in the pathogenesis of HD.

Follicular lymphomas' BCL-2/IgH junctions contain templated nucleotide insertions: novel insights into the mechanism of t(14;18) translocation.

The human t(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between BCL-2 and D(H)/J(H) gene segments during V(D)J recombination in early B cells. De novo nucleotides are found inserted in most breakpoints and have been thus far interpreted as nontemplated N region additions. In this report, we have analyzed both direct (BCL-2/J(H)) and reciprocal (D(H)/BCL-2) breakpoints derived from 40 patients with follicular lymphoma with t(14;18). Surprisingly, we found that more than 30% of the breakpoint junctions contain a novel type of templated nucleotide insertions, consisting of short copies of the surrounding BCL-2, D(H), and J(H) sequences. The features of these templated nucleotides, including multiplicity of copies for 1 template and the occurrence of mismatches in the copies, suggest the presence of a short-patch DNA synthesis, templated and error-prone. In addition, our analysis clearly shows that t(14;18) occurs during a very restricted window of B-cell differentiation and involves 2 distinct mechanisms: V(D)J recombination, mediating the breaks on chromosome 14 during an attempted secondary D(H) to J(H) rearrangement, and an additional unidentified mechanism creating the initial breaks on chromosome 18. Altogether, these data suggest that the t(14;18) translocation is a more complex process than previously thought, involving the interaction and/or subversion of V(D)J recombination with multiple enzymatic machineries.