RAG1/2 expression in follicular lymphoma marks candidate precursor cells of high-grade B cell lymphoma with MYC and BCL2 rearrangements

Abstract High-grade B cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) represent an infrequent, life-threatening clinical entity with poor prognosis. Recent advances, including novel chemotherapy regimens1, antibody-drug conjugates2, and CAR-T cell therapies3, have shown promise, yet durable disease-free survival remains elusive for most patients. A better understanding of the mechanisms underlying HGBCL-DH-BCL2 onset is key to the development of personalized therapies or integration of molecularly targeted interventions into existing regimens. Another potentially life-saving strategy involves anticipating HGBCL-DH-BCL2 emergence by studying the biology of follicular lymphoma (FL) that often precedes transformation. Identifying cell-intrinsic and/or microenvironment-associated molecular and spatial features specifically linked to FL evolution into HGBCL-DH-BCL2 may enable discovery of clinically relevant biomarkers predictive of transformation risk and alert on the presence of putative HGBCL precursor cells already present within FL. We have recently shown that HGBCL-DH-BCL2 frequently exhibit B cell receptor (BCR) silencing4. This phenotype is often accompanied by re-expression of the RAG1/2 recombinases, contributing to antigen receptor revision and irreversible BCR extinction4. Notably, we have provided evidence for RAG activity to be causally linked to HGBCL-DH-BCL2 outgrowth via IGL::MYC, t(8;22) translocations, postulating recombinase reactivation in the precursor cell as a key pathogenetic event4. To identify such precursor cells, we analyzed 10 FL cases for which RAG1/2 expression had already been measured in their metachronous HGBCL-DH-BCL2 counterpart4. FL specimens were paired with six RAG-positive HGBCL-DH-BCL2cases, while the remaining four paired with RAG-negative ones. On the same FFPE section, we combined RNA scope to detect RAG1/2 transcripts with DNA FISH for t(14;18) to spatially map RAG-expressing (pre)malignant B cells within FL. Also, RAG1/2 mRNA detection was matched with immunohistochemistry for LMO2 to assess the relation of RAG+ cells to the germinal center reaction. In a subset of cases, we compared Immunoglobulin (IG) light chain isotype usage between metachronous lymphomas to assess ongoing RAG-dependent receptor editing. RAG1/2 transcripts were detected in a distinct fraction of LMO2+BCL2+ t(14;18)+ cells in five of six (83%) FL cases anticipating RAG-positive HGBCL-DH-BCL2.An additional FL case displayed a sizeable pool of RAG1/2-expressing cells, despite its transformed counterpart failed to display detectable recombinases expression in the second bioptic sample. Three of the four RAG-negative HGBCL-DH-BCL2 cases mirrored this phenotype in their respective FL precursors. Building on our recent work showing that RAG-positive HGBCL-DH-BCL2 cases can exhibit IG light chain isotype inclusion4, we show that in two such cases, co-expression of IGK and IGL light chains is already present in the preceding FL, indicating early onset of antigen receptor revision. Altogether, in situ studies of paired FL and HGBCL-DH-BCL2 specimens identified for the first time a distinct subset of RAG-expressing, t(14;18)+ LMO2+ germinal center-like B cells in FL specimens that later progressed to recombinase-positive HGBCL-DH-BCL2. Given the causal link between RAG expression and IGL::MYC rearrangements caused by aberrant VJ recombination in a subset of HGBCL-DH-BCL24, we propose that RAG-expressing cells in FL represent strong candidates for HGBCL-DH-BCL2 precursor cells. References Melani C, Lakhotia R, Pittaluga S, et al. Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma. N Engl J Med. 2024;390(23):2143-2155. Schneider M, Nasta SD, Barta SK, et al. Analysis of Histologic, Immunohistochemical and Genomic Features of Large B Cell Lymphoma Tumors May Predict Response to Polatuzumab Vedotin Based Therapy in Patients With Relapsed/Refractory Disease. Clin Lymphoma Myeloma Leuk. 2025;25(1):45-51. Phina-Ziebin X, Bachy E, Gros FX, et al. Outcome of high-grade B-cell lymphoma compared with other large B-cell lymphoma after CAR-T rescue: a DESCAR-T LYSA study. Blood Adv. 2025;9(10):2500-2510. Varano G, Lonardi S, Sindaco P, et al. B cell receptor silencing reveals origin and dependencies of high-grade B cell lymphomas with MYC and BCL2 rearrangements. Blood Cancer Discov. 2025.