Polymerase chain reaction for detection of BCL1-MTC::IGHJ junction in mantle cell lymphoma

Key Figure.

(A) Diagram of BCL1-MTC and CCND1. cen, centromere; tel, telomere. (B) Agarose gel electrophoresis of PCR products representing BCL1-MTC::IGHJ junctions (arrows). The IGHJ segments rearranged with BCL1-MTC in each case are indicated at the bottom. The larger bands of lower density represent PCR products that extend to the next downstream IGHJ segments. (C) Nucleotide sequences of BCL1-MTC. The breakpoints in cases 1 to 10 are indicated by arrowheads. Bold letters refer to MTC determined by Nadeu et al. (GRCh38: 69532039–69532128).⁴

Mantle cell lymphoma (MCL) accounts for 2.0% of malignant lymphomas in Japan.¹ The majority (>95%) of MCL cases have a t(11;14)(q13;q32) translocation (Figure 1), the breakpoint of which on 11q13 was cloned by Tsujimoto et al. in 1984, using DNA probes of the IGH gene, and named BCL1 (B-cell leukemia/lymphoma 1).² Later, in 1991, it was revealed that the CCND1 gene encoding cyclin D1, then called PARD1 (parathyroid adenomatosis 1), was the target gene of t(11;14)(q13;q32);³ CCND1 is located 109 kb downstream or on the telomere side of BCL1, which is now called MTC (major translocation cluster) (Key Figure A).⁴

Figure 1.

(A) G-banding karyotype of case 10. The arrows indicate t(11;14)(q13;q32). The long arm of chromosome 3 telomeric to q21 is translocated to the short arm of chromosome 9 at p22, resulting in tetrasomy of 3q21→qter (asterisks). The karyotype is 46,XX,der(1)t(1;15)(p22;q11),add(6)(q11),der(9)t(3;9)(q21;p22)×2,t(11;14)(q13;q32),add(13)(q22),−15,add(17)(q25),add(21)(q22),min. (B) Metaphase FISH using Vysis IGH/CCND1 XT Dual Fusion FISH Probe Kit. Chromosomes 11 and 14 are labeled with the red-colored CCND1 and green-colored IGH probes, respectively, and der(11) and der(14) chromosomes are labeled with yellow signals representing the IGH::CCND1 fusion gene. Diagrams of the probes are presented at the bottom.

On the other hand, breakpoints on the IGH side of t(11;14)(q13;q32) almost always occur at 5′ of an IGHJ gene segment. As a result of t(11;14)(q13;q32), the 11q13/CCND1 region fuses with 14q32/IGH in the opposite direction, placing CCND1 under the control of enhancer elements of IGH, thereby leading to deregulated overexpression of cyclin D1. Because cyclin D1 is not expressed in normal lymphocytes, immunohistochemical detection of the protein is key to the diagnosis of MCL (Figure 2).

Figure 2.

Histopathology of the lymph node biopsy in case 8. Nuclear cyclin D1 expression was confirmed by two anti-cyclin D1 antibodies, SP4 and P2D11F11. Magnification of the objective lens is shown in parentheses.

Here, we present polymerase chain reaction (PCR) amplification of the BCL1-MTC::IGHJ junction. Genomic DNA was extracted from clinical materials of MCL patients treated in our hospital. We performed PCR using primers designed for the downstream sequence of BCL1-MTC and consensus sequence of IGHJ according to the BIOMED-2 protocol.⁵ Among a total of 20 MCL cases tested, 10 (50%) were positive for amplification (Key Figure B). The PCR products, ranging between 200 and 300 bp in size, were subjected to the cycle sequencing reaction (BigDye^TM Terminator v3.1 Cycle Sequencing Kit; Thermo Fisher Scientific, Waltham, MA, USA), and then sequenced using SeqStudio^TM Genetic Analyzer (Thermo Fisher Scientific). The data obtained were applied to BLASTn programs to identify closely related sequences. The results showed that the BCL1-MTC–side breakpoints were distributed within the 90-bp region determined as MTC by Nadeu et al. (Key Figure C).⁴ The IGH-side breakpoints, on the other hand, were at IGHJ3 in one case, IGHJ4 in 7 cases, and IGHJ6 in 2 cases (Key Figure B, Figure 3). At the junctions, the 5′-side 3 to 17 nucleotides of each IGHJ were deleted and N segments of 3 to 16 nucleotides were inserted (Figure 3). These sequences mimicked the structure of D to J recombination of IGH, in line with the general concept that generation of t(11;14)(q13;q32)/IGH::CCND1 is erroneously mediated by the V(D)J recombination process of IGH.⁶

Figure 3.

Nucleotide sequences of the BCL1-MTC::IGHJ junctions in cases 1, 5, and 9. Vertical lines indicate nucleotide identity. N-nucleotides inserted at the junctions are boxed. The positions of the nucleotides are numbered according to GRCh38.

Currently, fluorescence in situ hybridization (FISH) applied to metaphase spreads and/or interphase nuclei is the most commonly used technique to detect t(11;14)(q13;q32)/IGH::CCND1 (Figure 1B). Because 11q13/CCND1-side breakpoints are distributed over a large region upstream of CCND1, including BCL1-MTC,⁴ and because those in variant translocations, i.e., t(2;11)(p11;q13)/IGK::CCND1 and t(11;22)(q13;q11)/IGL::CCND1, involve areas downstream of the gene,^4,7 the CCND1 XT FISH probe included in Vysis IGH/CCND1 XT Dual Fusion FISH Probe Kit (Abbott Laboratories, Abbott Park, IL, USA) spans an approximately 942-kb region on 11q13/CCND1 (Figure 1B) ( https://www.molecularcatalog.abbott/int/en/Vysis-IGH-CCND1-XT-DF-FISH-Probe-Kit). In contrast, PCR of BCL1-MTC::IGHJ junctions can only detect 40–50% of cases with t(11;14)(q13;q32)/IGH::CCND1.⁵ Nevertheless, as the sensitivity of PCR ranges between 10⁻³ and 10⁻⁴,⁵ the method can be applied to not only diagnosis of MCL but also monitoring of the disease after treatment in PCR-positive cases. When choosing BCL1-MTC::IGHJ PCR as a diagnostic test for MCL, we must be aware of these advantages and disadvantages of the test compared with FISH.

REFERENCES

• 1)   Chihara  D,  Ito  H,  Matsuda  T, et al. Differences in incidence and trends of haematological malignancies in Japan and the United States. Br J Haematol 2014;164:536-545.
• 2)   Tsujimoto  Y,  Yunis  J,  Onorato-Showe  L, et al. Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation. Science 1984;224:1403-1406.
• 3)   Motokura  T,  Bloom  T,  Kim  HG, et al. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature 1991;350:512-515.
• 4)   Nadeu  F,  Martin-Garcia  D,  Clot  G, et al. Genomic and epigenomic insights into the origin, pathogenesis, and clinical behavior of mantle cell lymphoma subtypes. Blood 2020;136:1419-1432.
• 5)   van Dongen  JJ,  Langerak  AW,  Brüggemann  M, et al. 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. Leukemia 2003;17:2257-2317.
• 6)   Küppers  R,  Dalla-Favera  R. Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene 2001;20:5580-5594.
• 7)   Maekawa  F,  Kishimori  C,  Nakagawa  M, et al. Truncation of 3′ CCND1 by t(11;22) leads to negative SP4 CCND1 immunohistochemistry in blastoid mantle cell lymphoma. Blood Adv 2021;5:61-65.