DETECTION OF CLONAL IGH GENE REARRANGEMENT IN PLASMA CELL-FREE DNA (cfDNA) IN PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA BY SANGER SEQUENCING
Main Article Content
Abstract
Objective: To identify monoclonal immunoglobulin heavy chain (IGH) gene rearrangements in plasma cell-free DNA (cfDNA) in patients with diffuse large B-cell lymphoma (DLBCL).
Methods: A cross-sectional descriptive study of 31 patients diagnosed with DLBCL at the Blood Transfusion Hematology Hospital, Ho Chi Minh City, from October 2024 to September 2025. Peripheral blood collected at diagnosis, before treatment, was used for plasma separation and cfDNA extraction. IGH gene rearrangements were analyzed by multiplex fluorescent PCR with primers targeting the VH (FR1/FR2/FR3) and JH regions, followed by 2% agarose gel electrophoresis. Positive samples underwent capillary electrophoresis to determine clonality (monoclonal or polyclonal), and monoclonal cases were analyzed by Sanger sequencing to determine the characteristic VH–DH–JH patterns.
Results: IGH gene rearrangements were detected in 22/31 cases (71%) when all three VH regions (FR1/FR2/FR3) were analyzed simultaneously, and capillary electrophoresis identified monoclonal IGH rearrangements in 11/31 cases (35.5%). Sanger sequencing characterized specific VH–DH–JH rearrangements in 10/31 cases (32.2%); VH3 (50%) and VH4 (30%) were the predominant VH families, while DH6 (50%), JH1 (30%), JH5 (30%), and JH6 (30%) were the most frequent DH and JH segments.
Conclusions: Monoclonal IGH gene rearrangements can be detected in plasma cfDNA from patients with DLBCL using multiplex F-PCR combined with capillary electrophoresis and Sanger sequencing. These findings suggest the feasibility and potential of cfDNA as a molecular marker for supporting differential diagnosis and monitoring minimal residual disease (MRD) after treatment in patients with DLBCL.
Keywords
Diffuse large B-cell lymphoma, IGH gene rearrangement, Sanger sequencing
Article Details
References
2. Poletto S, Novo M, Paruzzo L, Frascione PMM, Vitolo U. Treatment strategies for patients with diffuse large B-cell lymphoma. Cancer Treat Rev. 2022;110:102443.
3. He J, Wu J, Jiao Y, Wagner-Johnston N. “IGH gene rearrangements as plasma biomarkers in Non- Hodgkin’s lymphoma patients”. Oncotarget. 2011 Mar;2(3):178-85. doi: 10.18632/oncotarget.235. PMID: 21399237; PMCID: PMC3260819.
4. Corcoran RB, Chabner BA. Application of Cell-free DNA Analysis to Cancer Treatment. N Engl J Med. 2018 Nov 1;379(18):1754-1765. doi: 10.1056/NEJMra1706174. PMID: 30380390.
5. Bo J, Sun L, Wang W, Ma C, Li H, Zhao Y. Novel diagnostic biomarker for patients with Non-Hodgkin’s Lymphoma by IGH gene rearrangement. J Cancer Res Ther. 2016;12(2):903-908. doi:10.4103/0973-1482.157345.
6. Nguyễn Vũ Hải Sơn, Lai Kim Phương, Cao Sỹ Luân, Phan Thị Xinh, “Khảo sát tái sắp xếp gen IGH trên NB đa u tuỷ xương tại Bệnh viện Truyền máu Huyết học bằng kỹ thuật PCR,” Y học Việt Nam 505 (2), pp. 235-239, 2021.
7. Van Dongen JJM, Langerak AW, Brüggemann Monika, et al, “DesIGn 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 Acion BMH4-CT98-3936,” Leukemia, 17 (12), pp. 2257-2317, 2003.
8. Groenen, P.J.T.A., van Raaij, A., van Altena, M.C. et al. A practical approach to diagnostic Ig/TCR clonality evaluation in clinical pathology. J Hematopathol 5, 17–25 (2012).
9. Zhong L, Huang WF. Better detection of IG heavy chain and TCRγ gene rearrangement in plasma cell-free DNA from patients with non-Hodgkin Lymphoma. Neoplasma. 2010;57(6):507-11. doi: 10.4149/neo_2010_06_507. PMID: 20845988
10. Sebastián, E., Alcoceba, M., Balanzategui, A., et al (2012). Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype. The American journal of pathology, 181(5), 1879–1888.