High-Throughput Cloning of T(11;14) Breakpoints Outside the Major Translocation Cluster in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is characterized by the over-expression of cyclin D1 (CCND1) as a result of the chromosomal fusion of the CCND1 gene at 11q13 to the immunoglobulin heavy chain (IgH) locus at 14q32. Up to 50% of t(11;14) breakpoints in MCL map to the ~200bp major translocation cluster (MTC) near the CCND1 gene. MCL patients having MTC breakpoints can be easily monitored for minimal residual disease (MRD) using a sensitive genomic PCR assay that employs consensus IgH joining (J_H) and CCND1-MTC primers. The majority of MCL patients, however, have t(11;14) breakpoints that are scattered across a ~380kb region outside the MTC and that have therefore been difficult to clone and use for MRD monitoring. We recently described Translocation-CGH (TGH), a modification of array-CGH that utilizes tiling-density arrays to map the genomic breakpoints of balanced translocations (including IgH fusions to BCL2, MYC and CCND1 loci) to ~100bp resolution and simultaneously detects genomic imbalances. We now report the use of TGH on a series of 33 confirmed MCL cases with CCND1 translocations, including 10 with MTC breakpoints and 23 with non-MTC breakpoints. In 32 of 33 cases (97%), we demonstrate the fusion of CCND1 to a J_H segment. The remaining case shows a genomic gain that encompasses the CCND1 gene and whose centromeric end extends to the MTC, presumably reflecting duplication of a derivative chromosome that contains a CCND1 fusion at the MTC to an as yet unidentified partner locus. Sequence analysis of the 23 non-MTC breakpoints reveals distribution across a ~330kb genomic region that surrounds the MTC and extends to within 3kb of the CCND1 transcription start site. Nine (39%) of the 23 non-MTC breakpoints were localized to simple or interspersed DNA repeats, including six (26%) in LINE1 elements, which comprise 11.7% of the CCND1 breakpoint region, and one each in LTR-ERVL, DNA-AcHOBO and simple-(GGAA)_n repeat elements. No breakpoints were identified within SINE/Alu repeats, which comprise 11.0% of the breakpoint region. The mechanism underlying the possible preference for LINE1 elements in CCND1 translocations (p=0.045, exact binomial calculation) is unknown and additional studies will be required to elucidate it. Preliminary sequence analysis of the der(14)-derived J_H-CCND1 junctions and the reciprocal der(11) breakpoints shows features similar to those described previously for MTC breakpoints. Interestingly, two of the non-MTC breakpoints are 1bp and ~170bp from previously identified breakpoints in the MCL cell lines MO1094 and Granta 519, respectively. However, we found no convincing evidence for ‘minor’ translocation clusters like those identified at the BCL2 locus in a subset of t(14;18)-positive lymphomas. In summary, the current study establishes the usefulness of TGH for identifying widely-scattered non-MTC breakpoints, which will enable the design of highly-sensitive and patient-specific genomic PCR assays for MRD monitoring in the vast majority of MCL patients.