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

Abstract

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 J H 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/J H 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/J H rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/J H 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/J H junctions harbored D H segments in their N regions indicating that bcl-1/J H rearrangements can occur in a later stage of B cell ontogeny during which the complete V H to D H -J H joining or V H -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.

Molecular remission is an independent predictor of clinical outcome in patients with mantle cell lymphoma after combined immunochemotherapy: a European MCL intergroup study

“…Samples were analyzed by t(11;14) (BCL1-IGH) PCR and IGH multiplex PCR as published to assess the clonal rearrangement. 32,33 Gene scanning and sequence analyses were performed on an ABI PRISM 377 automated sequencer (Applied Biosystems). Sequencing of clonal rearrangements for allele-specific RQ-PCR was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems).…”

Phase II study of a high-dose ifosfamide-based chemotherapy regimen with growth factor rescue in recurrent aggressive NHL. High response rates and limited toxicity, but limited impact on long-term survival

“…We hypothesized that increased dose-intensity might increase the rate of partial and complete remissions, and ultimately result in improvement in long-term outcome after autologous transplantation. [15][16][17][18][19] With filgrastim and prophylactic antibiotics, we were able to administer this regimen at 4-week intervals. Myelosuppression was profound, but relatively short.…”

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936

“…139,140 In nearly half of MCL cases (41%), the breakpoints were clustered within an 85 bp region that was referred to as the major translocation cluster region, BCL1-MTC. 137,141,142 In most, if not all, cases of MCL the break at the IGH locus located at 14q32 involves the JH genes juxtaposing the IGH-Em enhancer to chromosome 11q13 sequences, and consequently resulting in the transcriptional activation of the cyclin D1 gene. 143 Cyclin D1 together with CDK4 phosphorylates (and inactivates) pRB and allows for progression through the G1 phase of the cell cycle.…”

“…Many groups have developed PCR-based assays to detect the BCL1/JH breakpoints, in general using a consensus JH primer in combination with primers in the BCL1-MTC region that were all located in a region of 392 bp. 145,146 Breaks within the BCL1-MTC region can occur up to 2 kb downstream of the MTC region, but the majority of breakpoints are tightly clustered within an 85 bp segment, immediately downstream of the reported most 3 0 primer ('primer B' in Pott et al 141 Williams et al , 145 ). As breaks in this BCL1-MTC region account for only part of the breakpoints in the 11q13-BCL1 region in MCL cases (41%), the PCR-based strategy for t (11;14) seriously impairs the diagnostic capability with an high rate of false-negative results as compared to FISH.…”